Or Β-Hydroxy Γ-Lactam-Based HIV-1 Protease Inhibitors

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List of Papers

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

I Wu, X., Öhrngren, P., Ekegren, J. K., Unge, J., Unge, T., Wallberg, H., Samuelsson, B., Hallberg, A., Larhed, M. Two-Carbon-Elongated HIV-1 Protease Inhibitors with a Tertiary-Alcohol-Containing Transition-State Mimic. Journal of Medicinal Chemistry, 2008, 51, (4), 1053-1057.

II Öhrngren, P., Wu, X., Persson, M., Ekegren, J. K., Wallberg, H., Vrang, L., Rosenquist, A., Samuelsson, B., Unge, T., Larhed, M. HIV-1 Protease Inhibitors with a Tertiary Alcohol Containing Transition-State Mimic and Various P2 and P1' Substituents. MedChemComm, 2011, 2, (8), 701-709.

III Wu, X., Öhrngren, P., Joshi, A. A., Trejos, A., Persson, M., Arvela, R. K., Wallberg, H., Vrang, L., Rosenquist, Å., Samuelsson, B., Unge, J., Larhed, M. Synthesis, X-ray Analy- sis, and Biological Evaluation of a New Class of Stereo-Pure -Lactam-based HIV-1 Protease Inhibitors. Submitted

IV Öhrngren, P., Fardost, A., Russo, F., Schanche, J. S., Fagrell, M., Larhed, M. Evaluation of a Novel Non-Resonant Microwave Reactor for Continuous-Flow Applications. Submitted

Reprints are made with permission from the publishers.

Contents

1 Introduction ...... 11 1.1 Human Immunodeficiency Virus and Acquired Immune Deficiency Syndrome ...... 11 1.1.1 Introduction to HIV/AIDS ...... 11 1.1.2 The Replication Cycle of HIV ...... 13 1.1.3 Antiviral Drug Targets and Antiretroviral Therapies ...... 14 1.1.4 HIV-1 Protease and HIV Protease Inhibitors ...... 18 1.1.5 HAART/ART ...... 22 1.2 Tools in Medicinal Chemistry ...... 23 1.2.1 Microwave-Assisted Organic Synthesis ...... 23 1.2.2 Organic Reactions in Continuous-Flow Systems ...... 26 2 Aim of the Present Study ...... 29 3 Introduction of a Novel Tertiary-Alcohol-Containing Scaffold and Evaluation of P1' Substituents ...... 31 3.1 Chemistry ...... 32 3.2 Biological evaluation ...... 36 3.3 X-ray analysis ...... 39 4 Variations of the P2–P3 Moiety ...... 41 4.1 Chemistry ...... 41 4.2 Biological evaluation ...... 44 4.3 X-ray evaluation ...... 47 5 -Hydroxy -Lactams as Central Moiety in HIV-1 Protease Inhibitors ... 51 5.1 Chemistry ...... 51 5.2 Biological evaluation ...... 58 5.3 X-ray evaluation ...... 62 6 Non-Resonant Applicator for Microwave-Assisted Organic Synthesis in a Continuous Flow System ...... 65 6.1 The CF-MAOS Instrument ...... 65 6.2 Evaluation of Instrument Performance ...... 68 6.3 Evaluation of Model Reactions ...... 70 7 Concluding Remarks ...... 77

Acknowledgments...... 79 References ...... 83

Abbreviations

[bmim]BF4 1-butyl-3-methyl-imidazolium tetrafluoroborate 9-bbn 9-Borabicyclo[3.3.1]nonane Ac acetyl AIDS acquired immune deficiency syndrome Arg argenine ART anti-retroviral therapy Asp aspartic acid ATZ atazanavir BOC tert-butyloxycarbonyl bp boiling point CA capsid protein CC50 inhibitor concentration reducing the cell proliferation with 50% CF continuous-flow CF-MAOS continuous-flow–microwave-assisted organic synthesis CRI co-receptor inhibitor or entry inhibitor DCM dichloromethane DME 1,2-dimethoxyethane DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone DMSO dimethyl sulphoxide EC50 inhibitor concentration reducing viral cytopatic effect with 50% EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride FDA U.S. Food and Drug Administration FI fusion inhibitor Gly glycine gp glycoprotein HAART highly active antiretroviral therapy HIV human immunodeficiency virus HOBt 1-hydroxybenzotriazole HPLC high performance liquid chromatography HTLV human t-cell leukaemia–lymphoma virus Ile isoleucine IN integrase INI integrase inhibitor IS internal standard IUPAC International Union of Pure and Applied Chemistry Ki inhibition constant

LAV lymphadenopathy-associated virus LDA lithium diisopropylamide Leu leucine MA matrix protein MAOS microwave-assisted organic synthesis ME 1,2-dimethoxyethane MW microwave NC Nucleocapsid protein NMM N-methylmorpholine NMP N-methylpyrrolidone NNRTI non-nucleoside reverse transcriptase inhibitor NRTI nucleoside reverse transcriptase inhibitor NtRTI nucleotide reverse transcriptase inhibitor PDB Protein Data Bank Ph phenyl PI protease inhibitor PR protease Pro proline Py pyridiyl RNA ribonucleic acid RP reversed phase RT reverse transcriptase rt room temperature TBAF tetrabutylammonium fluoride TBAHS tetrabutylammonium hydrogen sulphate TBS tert-butyldimethylsilyl TBSOTf tert-butyldimethylsilyl trifluoromethanesulphonate TFA trifluoroacetic acid THF tetrahydrofuran Thr threonine TS transition state TsOH para-toluenesulphonic acid UNAIDS Joint United Nations Programme on HIV/AIDS Val valine WHO World Health Organization

1 Introduction

1.1 Human Immunodeficiency Virus and Acquired Immune Deficiency Syndrome

1.1.1 Introduction to HIV/AIDS The disease known today as acquired immune deficiency syndrome (AIDS) first came to light when an abnormal increase in reports of rare diseases such as Kaposi’s sarcoma and/or pneumonia caused by Pneumocystis carinii was noted in the early 1980s.1-4 These case reports were soon followed by the identification of the first patients suffering from AIDS, and the definition of the disease indicators.5 It was concluded that there must be a causative agent, probably a virus, assumingly transmitted via blood and/or sexual contact.6 Within three years of the first reports in 1983, research groups led by Montagnier and Gallo independently provided the first evidence of this hypothesis, identifying the transmissible agent as a virus,7,8 first called human T-cell leukaemia virus III (HTLV-III)8 or lymphadenopathy-associated virus (LAV).9 Later, the virus was cloned,10,11 the nucleotide sequence determined,12,13 and the virus causing AIDS14 was given the name used today, human immunodeficiency virus (HIV).15 Feorino et al. confirmed the transmission of the virus via blood transfusions, and concluded that many of the blood donors were asympto- matic, suggesting that HIV could exist in a latent phase.16 HIV belongs to the lentivirus subfamiliy of the retroviridiae17 and is a single-stranded RNA virus. The virus envelope has a conical shape, approximately 100 nm in length with a diameter of 20–60 nm,18 and carries two copies of the RNA sequence.19 HIV occurs in two main strains,7,8,20 denoted HIV-1 and HIV-2, identified in 1983 and 1986, respectively. HIV-1 has greater pathogenicity than HIV-2, is geographically more widespread and is the most common form among infected humans.21 HIV-1 can infect both resting and activated CD4+-T-lymphocytes, but needs activated cells to be able to replicate.22,23 After the identification of AIDS, cases were reported from all over the world, and AIDS was soon classified as a pandemic. HIV/AIDS affects mankind globally, although there are large variations in prevalence. The highest prevalence is in sub-Saharan Africa (with 68% of all cases of

11 HIV/AIDS and a prevalence above 15% in some regions), and in some areas of South-East and Central Asia. The latest report by WHO/UNAIDS estimates that today 33.3 million people are living with HIV/AIDS, that over 2.6 million new cases occur annually, and that there are almost 5000 AIDS- related deaths per day (Figure 1).24

Western & Eastern Europe Central Europe & Central Asia 820 000 1.4 million North America [720 000 – 910 000][1.3 million – 1.6 million] 1.5 million East Asia [1.2 million – 2.0 million] 770 000 Middle East & North Africa [560 000 – 1.0 million] Caribbean 460 000 [400 000 – 530 000] 240 000 South & South-East Asia [220 000 – 270 000] 4.1 million Sub-Saharan Africa [3.7 million – 4.6 million] Central & 22.5 million South America [20.9 million – 24.2 million] Oceania 1.4 million 57 000 [1.2 million – 1.6 million] [50 000 – 64 000]

Figure 1. Estimates of the number of people living with HIV/AIDS worldwide, November 2010.24 (Published with kind permission from UNAIDS.)

The WHO estimates that more than 60 million people have been infected, and that nearly 30 million people have died from AIDS since the beginning of the pandemic.25 When the disease was first identified, the expected survival time for a patient with HIV/AIDS was 2–5 years.26 With the development and introduction of the first antiviral drugs,27 the estimated lifespan started to increase and, at least in the developed countries, HIV/AIDS has become a manage- able, chronic disease.28,29 The number of people living with HIV/AIDS is still increasing, although this is partly due to the more efficient treatment regimen extending life expectancy. Encouragingly, UNAIDS also reported a decreasing number of newly infected people in many regions, in their latest report (Figure 2).24 A new trend, showing a reduction in the number of AIDS-related deaths in North America and Western and Central Europe, soon followed the introduction of the first drugs targeting HIV-1 protease (PR) in 1995,30 together with the introduction of the first non-nucleoside reverse transcriptase inhibi- tor31 (NNRTIs) the following year (Figure 2b).24 With increased global availability, the same trend is now being observed in most regions with, for example, Sub-Saharan Africa peaking in 2004 (Figure 2c), and the number of AIDS-related deaths per year has since fallen by 18%.24

12 a) 3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

b) c) 120 2

100 1.5 80

60 1

40 0.5 20

0 0

Figure 2. a) Number of newly infected (millions), 1990–2009; b) Number of AIDS- related deaths (thousands) in North America, Western and Central Europe, 1990– 2009; c) Number of AIDS-related deaths (millions) in Sub-Saharan Africa, 1990– 2009.24

1.1.2 The Replication Cycle of HIV The HIV-1 life cycle starts with a viral particle finding a target cell and attaching to its surface. HIV-1 expresses a surface protein, gp120, on the capsid with a high affinity for the host cell surface protein CD4. The CD4 is expressed mainly by CD4+-T-lymphocytes,9,32 but is also expressed by macrophages and in the human brain.33,34 Interaction with CD4 promotes binding to the chemokine receptors CXCR4 and/or CCR5.35-38 This complex causes conformational changes in the transmembrane protein gp41, which helps mediate the fusion of the virus particle and the host cell.35,39 Once inside the target cell the viral RNA is released in the cytoplasm and converted to DNA by reverse transcriptase (RT). During reverse transcrip- tion RT recombines the two RNA strands present in the HIV virion.40 This recombination process, which introduces frequent base substitution errors (~1/2000), goes a long way to explaining the rapid development of drug resistance.38,40-42 At this error rate, 5–10 errors can be introduced in every replication cycle.42 The viral pre-integration complex formed is transported to the cell nucleus and then integrated into the host genome by viral integrase (IN) as pro-viral DNA.35,43 When the host cell is activated the pro-viral DNA is transcripted to generate full-length viral mRNA and incompletely spliced

13 viral transcripts. These transcripts encode the major structural, regulatory and accessory proteins needed to form new viral particles.35 The major structural proteins are the Gag polyprotein precursor, including the matrix protein (MA) and the capsid protein (CA), the Gag–Pol polyprotein precursor (including RT, IN and PR) and Env polyprotein precursor (including gp160).18,39 In a process mainly controlled by the Gag and Gag–Pol polyproteins, new virus particles are assembled at the plasma membrane. Two copies of virus RNA are recruited as the genetic messengers in the new virus particle.35,39 After budding, the immature virus particle matures and becomes virulent when the PR cleaves Gag and Gag–Pol to give their active proteins (Figure 3).39 In total, the HIV genome includes nine genes (gag, pol, vif, vpr, vpu, env, rev, nef and tat) encoding 15 different proteins,35 among which RT, IN, PR and gp41 are today used as drug targets, as described below.

Figure 3. The replication cycle of HIV. Antiviral drugs have been developed targeting RT, PR and gp160. More recently, drugs have also been approved for CCR5 and IN. RNAseH, CXCR4, tat, nef and rev are other viral proteins under evaluation as drug targets.39,44 (Illustration from Flexner,44 reprinted with kind permission from Nature Reviews Drug Discovery.)

1.1.3 Antiviral Drug Targets and Antiretroviral Therapies There are several stages in the HIV life cycle that can be targeted in order to prevent the infection of a new cell or inhibit viral replication. Up until October 2011, the U.S. Food and Drug Administration (FDA) had approved

14 25 different substances, divided into six different classes,39,45 for use in the treatment of HIV.45 Being a retrovirus, HIV depends on RT to transform viral RNA into pro-viral DNA. This essential step was the first target in the search for an antiviral therapy.

Nucleoside Reverse Transcriptase Inhibitors The nucleoside reverse transcriptase inhibitors (NRTIs) were the first class of compounds found to be active against HIV.46 NRTIs need to be phosphorylated three times in the 5'-hydroxy position before they can be used by RT as building blocks in the synthesis of DNA. When phosphorylated, they compete with the natural building blocks in the active site.47 All NRTIs include either a 2',3'-dideoxyribose moiety or a derivative thereof, and thus lack the 3'-hydroxy group present in the natural substrates. The missing hydroxyl group leads to chain termination when incorporated into DNA.40,48 Resistance against NRTIs occurs mainly via impaired incorporation of the NRTI, or via the capability to remove the inserted NRTIs from the DNA sequence and replace them with the natural substrate.49 In 1987, 3'-azido-3'-deoxythymidine, better known as zidovudine or AZT,27 was the first drug to be approved by the FDA for the treatment of HIV. Today, this class of NRTIs has expanded to comprise six approved drugs (Figure 4).45

Figure 4. NRTIs approved for clinical use by the FDA.45

Non-Nucleoside Reverse Transcriptase Inhibitors Non-nucleoside reverse transcriptase inhibitors (NNRTIs) do not require phosphorylation to become activated,17 and differ from NRTIs in their mode of action. They bind to an allosteric binding site approximately 15 Å from the active site,28,50 causing a conformational change in the active site inhibiting its function.50 While NNRTIs have a reasonable toxicity profile and display a diversity of structure, they are still sensitive to resistance devevel- opment.17 The first NNRTI, nevirapine,31 was approved by the FDA in 1996, and four more have followed (Figure 5).45

15 O O S O HN HN NH F3C N N Cl O N N N N N H N O O H Nevirapine Delavirdine Efavirenz

NH CN NC CN NN N

H2N O N N N Br H H Etravirine Rilpivirine Figure 5. NNRTIs approved for clinical use by the FDA.45

Nucleotide Reverse Transcriptase Inhibitors Nucleotide reverse transcriptase inhibitors (NtRTIs) are sometimes included among the NRTIs.45 NtRTIs already carry one phosphonate group, and only require two phosphorylations to be able to act as building blocks for RT inducing chain termination.51 The phosphonate group included in NtRTIs has the advantage of not being cleavable by hydrolysis and is therefore more difficult to remove when incorporated into the growing DNA chain.51 NtRTIs show a better toxicological profile than most NRTIs.52 So far, only one NtRTI has been approved by the FDA (tenofovir, 2001,45,51-53 Figure 6) but other substances are undergoing clinical trials.

Figure 6. The NtRTI approved for clinical use by the FDA.45

Protease Inhibitors The introduction of the first protease inhibitor (PI), saquinavir,30 in 1995,45 heralded a new era in HIV treatment. PIs inhibit the HIV aspartic protease, which is responsible for cleaving the synthesised polyproteins into their active proteins and enzymes. Without this maturation process the virus particles produced would be non-infectious. HIV PR and PIs will be discussed in greater detail below (Section 1.1.4).

16 Fusion Inhibitors Fusion inhibitors (FIs) target and inhibit gp41, thereby blocking the fusion of the virus capsid with the host cell. Only one compound in this class of drugs has been approved by the FDA (enfuvirtide, 2003).45 Enfuvirtide (Figure 7) is a 36-amino-acid peptide consisting of amino acid residues 643–678 of gp160,54 which correspond to the amino acid residues 127–162 in gp41.55 Being a peptide, the compound is administered subcutaneously. Enfuvirtide is selective for HIV-1,54 but has a low genetic barrier and must be combined with other antiviral therapies to avoid resistance.56

Figure 7. The FI approved for clinical use by the FDA.45

Entry/Co-Receptor Inhibitors The compounds used to inhibit the chemokine receptors CXCR4 and/or CCR5 are called entry inhibitors or co-receptor inhibitors (CRIs).28,45,51 More attention has been devoted to finding a CCR5 inhibitor since the discovery that the naturally occurring 32-base-pair deletion (present in ~1% of all Caucasians) in the CCR5 gene generates resistance to infection by CCR5- dependent HIV strains.51,57,58 One CCR5 compound in this class of inhibitors has been approved by the FDA, maraviroc, 2007 (Figure 8).45,59

Figure 8. The CRI approved for clinical use by the FDA.45

17 Integrase Inhibitors Integrase catalyses both the essential 3'-processing of viral cDNA needed before strand transfer and strand transfer itself.60 Integrase Inhibitors (INIs) can act either on the 3'-processing or as inhibitors of strand transfer. Ralte- gravir, approved by the FDA in 2007 (Figure 9),45 acts as a strand transfer inhibitor, preventing the insertion of viral DNA into the host genome.61 Since there is no host target, INIs will not interfere with normal host cell activity and should be relatively non-toxic.28,43

Figure 9. The INI approved for clinical use by the FDA.45

1.1.4 HIV-1 Protease and HIV Protease Inhibitors

HIV-1 Protease HIV-1 PR is a two-fold symmetric dimer consisting of two identical subunits comprising 99 amino acids each. The PR is an aspartic protease with a conserved catalytic site, encompassing Asp25, Thr26 and Gly27 from each subunit, responsible for enzyme activity.62-64 When the natural unsymmetrical substrates (or a protease inhibitor) bind to the symmetrical PR, the conformation of the two monomers is changed and they lose their symmetry.64,65 The PR locates the proper cleavage sites more by recognition of peptide shape than amino acid sequence.66 Small variations between the different cleavage loci generate a strict order with different cleavage rates for each site.67 To be active, the PR must form dimers, which means that the activity of PR is dependent on the concentration of PR subunits. This arrangement prevents degradation of viral proteins before budding of new viral particles can occur.63 PR processes Gag and Gag–Pol polyproteins to active proteins and enzymes, including the structural capsid proteins (MA, CA, NC) and the enzymes (PR, RT, IN), with nine cleavage sites in total.66 This processing follows the general mechanism for aspartic proteases (Figure 10).68,69 A water molecule coordinating to Asp25 and Asp125 is activated by the negative charge carried by one of the Asps, and carries out a nucleophilic attack on the carbonyl next to the scissile bond in the substrate. This creates a tetrahedral intermediate. The amide nitrogen of the scissile bond is proto- nated and the reformation of the carbonyl and elimination of the amine cleaves the scissile bond (Figure 10).68,69

Figure 10. The mechanism of peptide bond cleavage by aspartic protease.68,69

Following the standard nomenclature, the amino acids on the C-terminal side of the scissile bond are denoted P1'-P2'-P3'…-Pn', and on the N-terminal side P1-P2-P3…-Pn. The corresponding pockets in the enzyme are denoted S1'- S2'-S3'…-Sn' and S1-S2-S3…-Sn on the C-terminal and N-terminal side of the scissile bond, respectively (Figure 11).70

Figure 11. The nomenclature commonly used to describe amino acid moieties and the corresponding enzyme pockets.70 The scissile bond is denoted by a dashed line.

The active site is covered by two so-called flaps, one from each monomer. The flaps are flexible and may be in the open or closed position. In the open position, the entry of substrates into the enzyme is promoted and when in the closed position, the processing of substrates is facilitated.65,71 Ile50 and Ile150 (from monomers one and two, respectively) co-ordinate a structural water molecule creating hydrogen bonds with the substrates,65,71 thus helping to position the substrates in the active site.69

Figure 12. HIV-1 protease displayed with the secondary structure as a ribbon cartoon with the flaps closed on top and the active site as the central cavity (PDB code: 2xye, Paper II)

19 X-ray analysis of the structures of HIV PR63,72 and PR co-crystallised with substrate-based inhibitors71 was carried out early (in 1989), and the knowledge derived from such studies has facilitated the development of antiviral drugs targeting PR.

HIV-1 Protease Inhibitors The PR was identified as a drug target as soon as its role in the viral replication cycle had been elucidated.73 PIs bind competitively to the active site of the PR and co-ordinates with the flaps in the closed position via a structural water molecule.74 All PIs available today, with the exception of tipranavir, have a peptidomimetic character, with the scissile bond replaced by a non- hydrolysable bond. The most commonly used transition-state (TS) mimic in HIV-1 PIs is the hydroxyethylene moiety.67 However, many other TS mimics have been tested in protease inhibitors (Figure 13).69

Figure 13. Examples of transition-state mimics.69

In 1995, the FDA approved the first antiviral compound in this class, saquinavir,30 and soon after ritonavir75 (1996), indinavir76 (1996) and nelfinavir77 (1997) were approved45 (Figure 14). This first generation of PIs were efficacious, but most of them suffered from low bioavailability and a short biological half-life, necessitating frequent dosage. These early PIs also caused severe adverse effects, especially gastrointestinal complications such as nausea, vomiting and diarrhoea, and metabolic disorders, e.g., hyperlipidaemia, lipodystrophy and insulin resistance.78,79 When used in monotherapy, resistant virus strains developed after a short time.74 The PIs were soon combined with NRTIs and/or NNRTIs in what has come to be known as highly active antiretroviral therapy, or HAART, a success story described in more detail in Section 1.1.5. The introduction of PIs had a considerable impact on survival rates and quality of life for those infected with HIV. The groundbreaking effect of PIs is well illustrated in the distinct change in the number of HIV/AIDS-related deaths since 1995 (Figure 2b).

Figure 14. PIs approved for clinical use by the FDA.45

The second generation of PIs entered the market in 1999 when amprenavir80 was approved. Amprenavir was followed by lopinavir81 (2000), atazanavir82 (2003), fosamprenavir83 (2003), tipranavir84 (2005) and darunavir85 (2006) (Figure 14). Atazanavir (ATZ) was the first PI with a once-daily dosing regimen, thereby reducing the tablet burden.86 Second-generation PIs showed improved pharmacokinetic properties and improved toxicity profiles compare to the first generation of PIs.78 Ritonavir was soon observed to be an efficient inhibitor of the enzyme cytochrome P450 3A4 (CYP3A4),87 known to metabolise many of the PIs.67

21 Administering a small amount of ritonavir together with other PIs boosted the effect of the co-administered PI by blocking major parts of the metabolism of the PIs.88 Today, ritonavir is mainly used as a booster for other PIs, seldom for its protease-inhibiting capacity.67 Several formulations with fixed dose combinations have been approved in an attempt to reduce tablet burden.45 At present, there are nine FDA-approved compounds in this class; saquinavir is only marketed as the saquinavir mesylate and amprenavir has been replaced by the prodrug fosamprenavir (Figure 14).45

1.1.5 HAART/ART The introduction of the PIs meant that a second target in the battle against HIV could be attacked. With the mono-therapies initially used, resistance was observed when using RT inhibitors or PIs.89 In combination therapy, NRTIs are combined with NNRTIs and/or PIs. A synergistic effect was achieved by combining drugs from different classes, lacking cross resistance and having non-overlapping toxicity profiles.17,90-94 This new treatment regimen was called HAART.90 Despite the good results obtained with PIs as part of HAART, the severe side effects commonly observed with early PIs prevented them from becom- ing first-line drugs in HAART. The most common combinations in early HAART regimes were instead two NRTIs together with a NNRTI. The development of NNRTI- and/or NRTI-resistant HIV strains and the introduction of new PIs, with a once-daily dosing regimen and improved effect profiles, has made the combination of a PI together with two NRTIs a more frequent first-line combination in HAART.28,94,95 Today, at least three different drugs are used in combination in HAART regimes.94 Two formulations with antiretroviral substances from different classes are available, in which different NRTIs, NNRTIs and a NtRTI are combined to reduce the tablet burden.45 The abbreviation ART, for antiretroviral therapy, is now more common, but it still refers to the same kind of combination therapy. With the ART currently in use, it is possible to suppress the HIV infection below today’s detection level (less than 50 copies viral RNA/mL blood), and thus minimise the development of resistant strains.89 Unfortunately, “undetectable” is not the same as “non-existent”. However, with ART the CD4 count is dramatically increased, normally to levels above 350 viral RNA copies/mL.89 Despite the available forms of treatment described above, there is a continuous need for new active anti-HIV drugs, i.e., compounds that are more efficacious against resistant strains, and which have improved pharmacokinetic and pharmacodynamic properties, and improved toxicity profiles.

22 1.2 Tools in Medicinal Chemistry

1.2.1 Microwave-Assisted Organic Synthesis The method used to heat chemical mixtures has remained almost the same since scientists first began to investigate chemical reactions. The use of open fire was abandoned in 1855 in favour of the controllable Bunsen burner,96 which was later replaced by heating plates, oil baths and electric mantles, but the principle, i.e. conductive heating, was still the same. A common problem with conductive heating is so-called wall effects. When using conductive heating there will always be a temperature gradient from the walls of the reactor towards the centre of the reaction mixture97-99 (Figure 15). The heating medium will have the highest temperature, followed by the walls of the reactor (equally hot in a perfect system), and a decreasing temperature gradient towards the centre of the reactor. Efficient stirring is used to minimise the effect, but it can often not be completely eliminated.

Figure 15. Heating profiles with conventional heating (left) and with microwave heating (right).98 (Published with kind permission from Molecular Diversity.)

The situation started to change some 25 years ago, when domestic microwave ovens were used for the first time in organic chemistry reactions. Microwave-assisted organic synthesis (MAOS) was born in the mid 1980s following reports by Gedye et al.100 and Giguere et al.101 in which they described the first MAOS reactions.

23 The frequency of microwaves ranges from 0.3 to 30 GHz ( = 100–1 cm), i.e. between IR radiation and radio frequencies.102 A frequency of 2.45 GHz ( = 12.2 cm) is most commonly used in both microwave applicators in organic chemistry and in domestic microwave ovens, to avoid interference with radar and telecommunications.103 When a reaction mixture is exposed to microwaves (MWs) the energy can be transferred to the reaction mixture in two different ways: dipolar polarisa- tion or ionic conduction. The oscillating electromagnetic field forces the dipoles in the medium to rotate and to try to align with the field, but at the frequencies commonly used the dipoles will not have time to align perfectly before the polarity of the field shifts again. This fluctuation forces the dipoles to move without being able to perfectly follow the field. This kinetic energy is then converted to heat by molecular friction and collisions giving rise to dielectric heating.104,105 Heat transfer by ionic conduction occurs due to the migration of ions with the electric field. Heat is again generated by molecular friction, and is mainly dependent on the size and charge of the ions.106 The dielectric loss ( '') describes how efficiently the microwave energy is transformed into heat, while the dielectric constant ( ') is a measure of how a molecule is affected (in this case polarised) by the electric field. The loss tangent (tan ) expresses the ability of a specific solvent or reaction mixture to be heated when exposed to microwaves. Tan is correlated to the dielectric loss and dielectric constant by the relation: tan = ''/ ' 102,107 and is dependent on the microwave frequency and temperature of the solvent. The loss tangent for a specific solvent or reaction will de facto change upon heating (Figure 16).102 Solvents with a high tan (>0.5) and low tan (<0.1) at room temperature, are classified as high- and low-microwave-absorbing solvents, respectively.97 When using microwaves as an energy source, a different heating profile is observed in the reactor compared to the use of conventional heating (Figure 15). The heating profile observed using microwaves is more uniform, and the walls are cooler than the reaction mixture, if a reactor of microwave- transparent material is used. Many of the undesirable wall effects observed with conventional heating are avoided when microwave radiation is applied, especially when rapid heating to high temperatures is required.98,99,106 The penetration depth of microwaves depends on the frequency of the radiation and the microwave-absorbing capacity of the material. In a standard microwave system (2.45 GHz) using common reactors made of borosilicate glass (microwave-transparent), the penetration depth in absorbing solvents is normally a few centimetres. The moderate penetration depth can be limiting when large volumes are to be heated.

Figure 16. Illustration of the temperature and frequency dependence of ' and '' exemplified by the dielectric properties of water at three temperatures. The frequency most commonly used (2.45 GHz) is shown by the dashed line.108 (Published with kind permission from Wiley-VCH.)

Purpose-built microwave ovens for organic chemistry reactions were described less than ten years after initial reports of microwave heating.109,110 Today, the microwave equipment used for organic chemistry reactions con- sists of highly dedicated and safe single- or multimode instruments.98,111,112 Single-mode microwave applicators are most commonly used for small-scale reactions (0.2–20 mL), whereas multimode applicators are used for larger- scale reactions. In domestic microwave ovens the microwaves are randomly distributed in the cavity, leading to regions of high and low energy density.99 This has a negative impact on reproducibility when heating small samples, and since the dedicated multimode applicators are based on the same technology, so-called mode stirrers are used to minimise these variations (Figure 17).97 The cavities in multimode applicators are larger than in single-mode applicators, allowing larger volumes or multiple reactions to be heated in parallel.97 In single-mode applicators the reaction vessel (reactor) is exposed to a continuous standing wave with well-defined characteristics (e.g. minimum and maximum field strengths). The well-defined characteristics of single- mode applicators, together with good temperature control, ensure a high degree of reproducibility (Figure 17).103,113 With the more efficient heating provided by microwave heating systems, the reaction times can often be shorted. When using sealed reactors in both single- and multimode applicators, solvents can be heated well above their atmospheric boiling point.113 This superheating makes it possible to shorten the reaction times even further. The rule of thumb states that the reaction time can be halved for every ten-degree increase in the reaction temperature.114 Efficient heating and reduced reaction times may provide energy savings, as well as savings in time and labour in drug development and production, and this technology should thus be investigated further.115-118

25 Microwave cavity (reactor illustrated in dashed line)

Magnetron

Magnetron Microwave cavity Rotating (reactor in dashed line) diffuser

Waveguide Microwavetransparent wall Magnetron Waveguide Figure 17. Schematic illustrations of a single-mode (left) and multimode (right) microwave applicator. Illustration modified from Ondruschka et al.119

In the novel continuous-flow (CF)-MAOS instrument developed in this work, and described in detail in Chapter 6, the microwaves are applied in a different fashion from the single- and multimode instruments currently available using a non-resonant applicator.

1.2.2 Organic Reactions in Continuous-Flow Systems Historically, the development of reaction vessels has been similar to that of heating. Until recently, lab-scale reaction vessels had changed little, being batch reactors of some kind, although some development has taken place over the years. The CF concept has mainly been applied on the industrial scale, and has not found its way into research labs until quite recently. Some examples were presented at the beginning of the 1970s, after which peptide synthesis in CF mode became more common.120-122 Several laboratory-scale CF reactors for synthetic chemistry have made use of micro-technology with flow channels of diameters less than 1 mm.123,124 The main advantage of CF systems is the high surface area to volume ratio, allowing relatively fast temperature changes.124-126 In many cases, reactions show better selectivity and produce higher yields when transferred from batch to CF systems.126,127 This could be explained, at least to some extent, by the constant reaction environment. In a batch reactor, the ratio between the different components changes continuously from the moment the reaction is started. In a CF system, the product and any side- products will be instantly transported away as fresh reactants enter the flow reactor.124 From the safety perspective, CF systems have the advantage of only heating a small volume at a time.123,127 This is beneficial when the reaction

26 contains highly toxic or explosive reagents, or when the reaction itself is highly exothermic. If the reaction becomes uncontrollable, only a small amount of substrate will be heated in the reactor, reducing the risk of a seri- ous accident. The possibility of scale-out is another advantage of CF systems. To increase the scale using a CF-system, the same system could simply be run for a longer time, or several similar systems could be run in parallel. This could be done without the costly re-optimisation normally necessary when increasing the scale to medium- or large-scale in batch syntheses.123 A disadvantage of CF microsystems (and to some extent most CF systems) is the need for particle-free reaction solutions. This is especially true in micro-systems where particles will clog the narrow channels.124 The majority of currently available CF systems employ either syringe pumps or HPLC pumps. HPLC pumps can produce higher pressures but are more sensitive to reagents than syringe pumps. Most commercial CF systems available today utilise conductive heating via heated oil,128 heated air,129 electric resistance130 or induction.131,132 The first report of a microwave- heated CF system was published by Strauss et al. in 1994.106,109 In more recent years, microwave-heated batch reactors have been modified to serve as CF-MAOS systems.133-135 These systems have been equipped with special reactors that fit inside the cavities of microwave batch instruments to allow heating with microwaves. Spiral flow cells made of borosilicate glass,136 u- shaped borosilicate glass loops,137,138 standard 10 mL vials filled with sand,139 glass bead reactors140 and micro-capillary flow cells141 have been used as reactors.

2 Aim of the Present Study

At the Division of Organic Pharmaceutical Chemistry, research is conducted in the fields of medicinal chemistry and microwave-assisted organic synthesis. The work presented in this thesis is part of ongoing research in both these areas.

One of the projects in the field of medicinal chemistry is concerned with identifying novel structures that can be used as HIV-1 protease inhibitors. The specific objectives in this part of my work were:

to develop novel scaffolds comprising a tertiary alcohol as part of the transition state mimic

to find alternatives to the commonly used indanol amide as the P2-P3 moiety

to make the protease inhibitor backbone more rigid to improve the protease inhibitor capacity

During the course of the above investigations, and as part of the project in microwave-assisted organic synthesis, a study was conducted to evaluate a novel non-resonance microwave applicator designed for continuous-flow applications.

3 Introduction of a Novel Tertiary-Alcohol- Containing Scaffold and Evaluation of P1' Substituents

Researchers at the Department of Medicinal Chemistry have been engaged in the development of novel HIV-1 PIs since the mid 1990s. Inspired by work by Lam et al.142 the first projects focused on symmetric and asymmetric cyclic HIV-1 PIs.143-145 Following the approval by the FDA of linear HIV-1 PIs in 1995, the focus shifted to linear inhibitors. Especially the launch of indinavir in 1996 and atazanavir in 2003 presented structural fragments of high interest. A TS mimic containing a tertiary-alcohol was created by relocating the commonly used secondary hydroxyl group present in TS mimics, e.g. hydroxyl ethylene, statin and hydroxyethylamine (Figure 13). The focus of research at the Department has since shifted towards developing HIV-1 PIs with this new tertiary-hydroxyethyl hydrazide as a TS mimic. The first HIV-1 PI scaffold with this novel TS mimic was presented by Ekegren et al. in 2005, using a one-carbon spacer between the tertiary alcohol and the hydrazide group (Figure 18, A).146-148 In the present work, the length of the inhibitor backbone spacer was extended to three carbon atoms in an effort to optimise binding to the aspartic catalytic site (Paper I and Figure 18, 13v).

Figure 18. Examples of novel HIV-1 PIs with a one-carbon spacer147 (A), and a three-carbon spacer (compound 13v in Paper I).

31 3.1 Chemistry The HIV-1 PIs with three-carbon scaffolds were synthesized from two primary parts, the non-prime and the prime moiety. Commercially available (S)-2-hydroxy-3-phenylpropionic acid (1) was used as the starting point for the synthesis of the non-prime side (Scheme 1). The alcohol and carboxylic acid functionalities were initially protected using 2,2-dimethoxypropane.149 A Michael addition of 2 with methyl acrylate using lithium diisopropylamide (LDA) gave 3, in which the tertiary centre had formed. Trifluoroacetic acid (TFA)-mediated deprotection of 3 caused intramolecular lactone formation with the methyl ester. Subsequent amide coupling with (1S,2R)-1-amino-2- indanol gave 4 as a mixture of diastereomers. The diastereomers were separated on a silica flash column giving (R)-4 and (S)-4 in good yields (Scheme 1). The absolute configuration of the quaternary carbon in (S)-4 was determined by X-ray crystallography (Figure 19a).

Scheme 1. Synthesis of the non-prime side building block (R)-6

Reagents and conditions: (a) pyridinium p-toluenesulphonate, chloroform, 70 °C, 97% isolated yield; (b) LDA, THF, 78 °C, 54% isolated yield; (c) i. TFA, H2O, 80 °C. ii. EDC, HOBt, DCM, rt, 41% isolated yield (R)-4, 46% isolated yield (S)-4, separated by silica flash column chromatography; (d) TBSOTf, Et3N, DCM, 0 °C to rt, 92% isolated yield; (e) i. LiBH4, diethyl ether, 0 °C. ii. Trimethylacetyl chloride, pyridine, rt. iii. TBSOTf, Et3N, b DCM, 0 °C to rt. iv. LiBH4, diethyl ether, rt, 73% isolated yield. (S)-6 was prepared from (S)-4 according to a slightly modified procedure, see Supporting information, Paper I.

The indanol moiety in the (R)-4 diastereomer, exhibiting the preferred stereochemistry identified in previous studies64,146-148 (Figure 19b), was protected using tert-butyldimethylsilyl trifluoromethanesulphonate (TBSOTf), Scheme 1.

32 a) b)

Figure 19. a) ORTEP plot to determine the stereochemistry of (S)-4. b) Note that when comparing the series of inhibitors with one- and three-carbon spacers, the absolute configuration of the quaternary carbon changes due to changes in assigned priority according to the sequence rule in IUPAC guidelines,150,151 despite the fact that the geometric configuration is identical.

With the indanol amide hydroxyl group protected by a tert-butyldimethylsilyl group (TBS), the indanol amide lactone, (R)-5, was reduced to the corresponding diol using lithium borohydride. This was followed by a series of protection and deprotection reactions. In the previous series146-148 the primary alcohol was directly oxidised to the aldehyde and reductively aminated with the prime side moiety. Applying the same methodology to the three-carbon scaffold only regenerated the favourable -lactone (5). In the novel three-carbon series, the primary alcohol had to be protected with trimethylacetyl chloride followed by protection of the tertiary alcohol using TBSOTf, after which selective deprotection with lithium borohydride gave the alcohol (R)-6 (Scheme 1). The previously successfully used P2' L-tert-leucine and P3' methyl carb- amate were chosen as building blocks for the prime side element.82,146,147 L- tert-leucine, 7, was treated with methyl chloroformate under basic conditions to form 8. Subsequent amide coupling with tert-butyloxycarbonylhydrazine using 1-(3-dimethylamiopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM),152 followed by removal of the protective group gave 9 in good yield (Scheme 2).

Scheme 2. Synthesis of the hydrazide building bock 9

Reagents and conditions: (a) methyl chloroformate, 2 M NaOH, 1,4-dioxane, 60 °C, overnight, 96% isolated yield; (b) i. tert-butyloxycarbonylhydrazine, EDC, HOBt, NMM, ethyl acetate, 91% isolated yield. ii. 4 M HCl, 1,4-dioxane, rt, 1.5 h, 79% isolated yield.

33 To evaluate the influence of the P1' moiety, a diverse series of hydrazides, 11a–s, were synthesized from the corresponding alkyl-, aryl- and biaryl- benzaldehydes (10a–c, 10e–r) or the halobenzyl compounds 12d and 12s. Reductive amination was used to couple aldehydes 10a–c and 10e–r with hydrazide 9 to form prime side moieties. Prime side 11d and 11s were synthesized from the 4-bromobenzyl bromide (12d) and 4-iodobenzyl bromide (12s), respectively, using hydrazine hydrate,153 followed by amide coupling with the free acid, 8, using EDC and HOBt, according to a previously described procedure82,146 (Scheme 3).

Scheme 3. Synthesis of hydrazides 11a–s

Reagents and conditions: (a) TsOH, NaBH3CN, THF, rt, 6–85% isolated yield; (b) i. 4- bromobenzylbromide (12d) or 4-iodobenzylbromide (12s), hydrazine hydrate, EtOH, 0 °C to room temp. ii. 8, EDC, HOBt, DCM, rt, giving 11d and 11s in 20% and 15% isolated yield, respectively.

The non-prime side moiety (R)-6 was oxidised with Dess–Martin periodinane to the corresponding aldehydes. Thereafter, reductive amination with prime side moieties and deprotection of the two alcohols gave inhibitors 13a–s in 19–91% isolated yield (Scheme 4). The inhibitor with 4-bromo- phenyl hydrazine as P1' was also isolated as the di- and monoprotected compounds 14 and 15 to evaluate the importance of the hydroxyl groups.

34 Diprotected compound 14 was used as the starting material in cross-coupling functionalisation reactions applying microwave-assisted batch synthesis.

Scheme 4. Synthesis of inhibitors 13a–w, 14, 15 and 18

O R a O (R)-6 11a-s H N N N O H OH N OH H O

13a-s Br

O b O (R)-6 11d H N N N O H OR'' N H OR' O

14 R' = TBS, R'' = TBS c 15 R'=H,R''=TBS R=

R t

u O d O N H 14 Ar B(OH)2 N N N O v N 16t-w H OH N OH H O w 13t-w S

O O O O O e f H (R)-6 N OH N N N O H OTBS H OH N TBSO OH H O 17 18 Reagents and conditions: (a) i. Dess–Martin periodinane, DCM, rt. ii. 11a–s, Na(OAc)3BH, acetic acid, THF, rt. iii. TBAF (10 equiv), THF, rt, 19–91% isolated yield; (b) i. Dess–Martin periodinane, DCM, rt; ii. 11d, Na(OAc)3BH, acetic acid, THF, rt, 48% isolated yield; (c) TBAF (1 equiv), THF, rt, 75% isolated yield; (d) i. arylboronic acid 16t–w, K2CO3, Herrmann’s palladacycle, [(t-Bu)3PH]BF4, DME/H2O, 120 °C or 130 °C, 20 min. ii. TBAF (10 equiv), THF, rt, 61–80% isolated yield; (e) i. Dess-Martin periodinane, DCM, rt, 1 h. ii. 2- methyl-2-butene, t-BuOH, H2O, NaClO2, NaH2PO4, rt, 6 h, 99%. (f) i. 11d, EDC, HOBt, DCM, rt; ii. TBAF (10 equiv), THF, rt, 39% isolated yield.

Palladium-catalysed Suzuki–Miyaura cross-coupling154-157 of arylboronic acids 16t–w with 14 using Herrmann’s palladacycle158,159 as the palladium 160 precatalyst and the (t-Bu)3P-releasing salt [(t-Bu)3PH]BF4 in a 1,2-

35 dimethoxyethane (DME)/water mixture irradiated for 20 minutes at 120 °C or 130 °C gave inhibitors 13t–w in good yields (Scheme 4). The acid derivative 17 was produced by two-step oxidation of (R)-6, and after amide coupling with 11d and deprotection of the alcohols, the diacylhydrazine inhibitor 18 was obtained (Scheme 4).

3.2 Biological evaluation The antiviral activities of inhibitors 13a–w, 14, 15 and 18 were evaluated on HIV-1 PR161,162 and in an MT4 cell-based assay.162 The results are summarised in terms of Ki (for the enzyme-based assay) and EC50 (for the cell- based assay), together with toxicity measurements in MT4 cells expressed as CC50 values, see Table 1. Atazanavir, indinavir and the previously described inhibitor A147 with a one-carbon spacer, are included for comparison. As previously, the stereochemistry at the carbon comprising the tertiary alcohol was of great importance. (S)-13d exhibited little or no inhibition and was 100 times less potent than the corresponding (R)-isomer. When both alcohols were not available for hydrogen bond interactions, as in 14 and 15, the antiviral activity was greatly reduced. No benefits were observed when an extra carbonyl was included to form a diacylhydrazine (18); in fact, the inhibitory capacity was reduced. With the hydroxyl groups deprotected and the (R)-configuration at the carbon comprising the tertiary alcohol, good inhibitors were identified, with Ki values of 2.4–11 nM. One exception was, however, the bulky 13p, which had a Ki of 24 nM. The most potent inhibitors in the series were the relatively hydrophilic heteroaromatic structures 13i, 13l–n, and 13u–13v (EC50 = 0.17–0.22). However, the differences throughout the series are small with at most a ten-fold difference in EC50 values between the active compounds. The cell permeability (Papp) and metabolic stability of compounds 13a, 13d, 13n and 13t–v were evaluated. Compounds 13d, 13n and 13t were -6 observed to have high permeability (Papp >20 × 10 cm/s) according to the Caco-2 assay,163 while 13u and 13v both showed intermediate permeability -6 -6 (20 × 10 cm/s > Papp >3 ×10 cm/s) and high stability (94% parent compound remaining), when incubated with human liver microsomes for 30 min at 37 °C.163 In comparison, indinavir and saquinavir have previously been found to have low permeability,164 while ritonavir164 and atazanvir146 have been shown to have intermediate permeability.

36 Table 1. HIV-1 antiviral activity and cytotoxicity of compounds 13a–w, 14, 15 and 18a

Compound R = Ki (nM) EC50 (µM) CC50 (µM) A 5.0 0.18 >10 13a 5.7 1.20 >10

13b 6.7 0.98 >10

13c 3.4 0.91 >10

13d 3.3 0.85 >10

(S)-13db 420 >10 >10

13e 2.9 1.20 >10

13f 4.9 0.98 >10

13g 6.8 1.80 >10

13h 2.8 0.47 >10

13i 2.4 0.22 >10

13j 3.6 0.50 >10

13k 11 0.88 >10

13l 3.3 0.17 >10

13m 3.6 0.19 >10

37 Table 1. Continued

Compound R = Ki (nM) EC50 (µM) CC50 (µM)

13n 2.3 0.21 >10

13o 3.5 0.51 >10

13p 24 1.00 >10

13q 2.3 0.48 >10

13r 5.5 0.60 >10

13s 4.5 0.82 >10

13t 7.3 0.78 7.5

13u 3.6 0.19 >10

13v 2.8 0.17 >10

13w 10 0.56 >10

14c 980 >10 >10

15d 190 >10 5.8

18e 120 >10 >10

IND165-167 - 0.52 0.041 - ATZ168 - 2.7 0.004 - a The Ki values of the novel structures were determined by two independent measurements. For biological activity determination methods and control of assay variability, see Paper I, Supporting information, pages S17-S18. b(S)-configuration at the tertiary alcohol. cBoth alcohols TBS-protected. dThe tertiary alcohol TBS-protected. eCentral diacylhydrazine unit.

38 3.3 X-ray analysis Inhibitors 13d and 18 were co-crystallised with HIV-1 PR (Figure 20). These novel inhibitors fitted well in the active site of HIV-1 PR, despite the three-carbon backbone scaffold. Inhibitor 13d formed five direct hydrogen bonds with the enzyme and three more via water molecules. The corresponding numbers of bonds for 18 were seven direct and five indirect bonds. Both 13d (2.9 Å) and 18 (2.9 Å) had one hydrogen bond to Asp25.

c) Br

O O H N N N O H OH N OH H O

Figure 20. Comparison of the overall conformations and binding patterns of the compounds 13d (a, PDB code 2uxz) and 18 (b, PDB code 2uy0). Inhibitor B with a one-carbon spacer is shown for comparison (c, PDB code 2cej).147 Hydrogen bonds are denoted by dashed green lines.

39 Both 13d and 18 lost one hydrogen bond with Asp125, compared to the previously published one-carbon tethered inhibitor B,147 which have two hydrogen bonds to the catalytic aspartic acids. The orientation of the P1' moiety in 13d differed from 18 and B, although the orientation in 13d was in better accordance with previous observations in other inhibitors in the same series as B,147 as well as in other series (Papers I, II and ref 169). The extra carbonyl in 18 can act as a hydrogen bond acceptor but in this configuration it will be repelled by the negatively charged Asp25 and Asp125 groups. The disadvantage from a binding point of view was also clearly reflected by the 40-fold increase in Ki for 18 compared with 13d (Table 1).

40 4 Variations of the P2–P3 Moiety

Although the first series with backbone-elongated inhibitors comprising a three-carbon scaffold (Paper I, Chapter 3) showed good inhibition in both enzyme- and cell-based assays, we sought to further elaborate the P2–P3 position. The (1S,2R)-1-amido-2-indanol, described in Paper I as well as in previously published series,146,147 is known to undergo rapid metabolic degeneration, primarily by benzylic oxidation.170,171 The aim of this part of the project was to find alternatives to the indanol P2–P3 moiety with improved, or at least the same, antiviral capacity. A small series of PIs with variations in the P1' side chain was also included (Paper II).

Figure 21. Schematic comparison of 13v (Paper I) and the inhibitors 23a–f and 25a–j (Paper II).

4.1 Chemistry Compound 3 was synthesized as described in Paper I and Section 3.1 from commercially available starting material. Subsequent heating of 3 with TFA at 80 °C overnight hydrolysed the dioxolane, and intramolecular -lactam formation gave 19 in excellent yield. The methylamide-derivatised amino acids 20a–e and TBS-protected L-tert-leucinol (20f) were used in peptide bond formation, mediated by EDC and HOBt,152 to give compounds 21a–f as mixtures of diastereomers. The diastereomers 21a–e were separated on a silica flash column and the (R)-derivatives were used in the following structural modifications. Note that compound 21f was carried forward as a diastereomeric mixture until the epimers could be separated by extensive RP-HPLC purification of the final compound, 23f.

41 Lactones 21a–f were opened by lithium borohydride and the same protection group methodology as described in Paper I (Section 3.1) was applied to form 22a–f. The primary alcohol in 22a–f was oxidised using Dess–Martin periodinane to form the corresponding aldehyde. Subsequent reductive amination coupled the aldehydes with the prime side moiety 11d146 (Section 3.1, Paper I) to form the inhibitors 23a–f after deprotection of the tertiary alcohol (Scheme 5).

Scheme 5. Synthesis of inhibitors 23a–f encompassing different P2 substituents

Reagents and conditions: (a) TFA, H2O, 80 °C, 95% isolated yield; (b) amino acid derivatives 20a–f (for R-groups see Table 2), EDC, HOBt, DCM, rt, 25–46% isolated yield ((R)-21a–f), 47% isolated yield ((S)-21a), diastereomers separated by flash chromatography; (c) i. LiBH4, diethyl ether, rt. ii. trimethylacetyl chloride, pyridine, rt. iii. TBSOTf, Et3N, DCM, 0 °C to rt. iv. LiBH4, diethyl ether, rt, 25–62% isolated yield; (d) i. Dess–Martin periodinane, DCM, rt. ii. 11d, Na(OAc)3BH, acetic acid, THF, rt. iii. TBAF, THF, rt, 3–81% isolated yield (Table 2).

Compound 23a was chosen for further optimisation of the P1' side. To be able to efficiently functionalise the P1' position through Suzuki–Miyaura cross-coupling reactions, the TBS-protected derivative, 24, was isolated before the final deprotection step in the synthesis of 23a and used as the starting material in the cross-coupling reactions (Scheme 6). Suzuki–Miyaura cross-coupling was performed in sealed reactors, by applying microwave heating in a single-mode applicator for 20 min at 120 °C. Complete conversion of the starting material was observed when 158 K2CO3 was used as base, Herrmann’s palladacyle as the palladium pre- 160 catalyst, a (t-Bu)3P-releasing salt [(t-Bu)3PH]BF4, and a DME/H2O mixture as solvent to give inhibitors 25a–e in moderate to good yields (Scheme 6 and Table 2).

42 Scheme 6. Microwave-assisted synthesis of inhibitors 25a–h

Reagents and conditions: (a) Suzuki–Miyaura cross-coupling; RB(OH)2, K2CO3, Herrmann’s palladacycle, [(t-Bu)3PH]BF4, DME/H2O, 120 °C, 20 min; (b) Sonogashira coupling; ethynyl pyridine, PdCl2(PPh3)2, CuI, Et3N, DMF, 130 °C, 60 min; (c) Heck alkynylation; ethynyl pyridine, PdCl2(PPh3)2, piperidine, H2O/acetone, 130 °C, 60 min or 140 °C, 30 min; (d) TBAF, THF, rt, giving 25a–h in 14–82% isolated yield (Table 3).

Sonogashira coupling chemistry172 was employed to elongate the P1' moiety 173 with ethynyl-pyridyl groups. A protocol with PdCl2(PPh3)2 and CuI microwave heated in a single-mode applicator for 60 min at 130 °C174 provided 25g in an isolated yield of 14%. Different protocols were evaluated147,175-177 and a copper-free protocol (a Heck alkynylation) inspired by a protocol reported by Shi and Zhang177 and Heck,178 was developed. Products 25f and 25h were synthesized in improved isolated yields of 30% and 29%, respectively (Scheme 6 and Table 3).

Scheme 7. Synthesis of inhibitors 25i–j

Reagents and conditions: a) i. Dess–Martin periodinane, DCM, rt. ii. 11h or 11n, Na(OAc)3BH, acetic acid, THF, rt. iii. TBAF, THF, rt, giving 25i and 25j in 67% and 75% isolated yields, respectively.

The P1' moiety could not be functionalised with the 2-pyridinyl and the thia- zolyl moieties via Suzuki–Miyaura cross-coupling due to the competing

43 protodeboronation155 and the lack of commercially available boronic acids at the time. To be able to introduce this type of moiety the methodology described in Paper I was used, starting from the corresponding aldehydes (Scheme 3, Scheme 7 and Table 3).

4.2 Biological evaluation

PIs 23a–f and 25a–j were evaluated regarding HIV-1 PR inhibition (Ki) and HIV-1 antiviral activity using a cell-based assay (EC50). The results are summarised in Table 2 and Table 3 below. The antiviral activity of PIs 23a–f clearly depended on the size of the P2 moiety. When the size of the P2 fragment was increased, as in 23b and 23e, higher concentrations were needed to achieve inhibition (Ki >50 nM). When the P2 moiety was reduced in size to a methyl, as in 23d, the inhibitor showed reduced activity, although not as much as in the case of 23b and 23e. The best inhibition was observed with the intermediate-sized P2 fragments tert-leucine (23a) and valine (23c), which exhibited Ki values of 6.2 and 2.7 nM, and EC50 values of 2.0 and 2.1 µM, respectively. As found in previous series, neither the TBS-protected inhibitor 24 nor the (S)-epimer of 23a inhibited the enzyme. Compound 23f was designed to conserve the hydrogen bonding pattern observed when using the amido- indanol P2–P3 fragment. However, compound 23f gave low inhibition of the enzyme (Ki = 130 nM) and no inhibitory effect in the cell-based assay (Table 2). Further optimisation of the P1' moiety did not significantly improve the antiviral activity of this class of inhibitors compared to 23a (Table 3). En- zymatic inhibition of the best inhibitor in the series, 25b, was almost equipotent to 13v, although it was slightly less potent in the cell-based assay (Table 3). P1' moieties extended with the ethynyl groups (25f–h) were tolerated but not beneficial. In this series, the 3-pyridyl-functionalised inhibitors 25b and 25g showed slightly better inhibition than their 2-pyridyl analogues (25i and 25f, respectively). In the cell-based assay, inhibitors 25a–f exhibited EC50 values around 1 µM. The greatest diversity was observed with the 4-ethynyl pyridiyl inhibitor 25h, which was almost ten times less potent than the best inhibitors in the series (Table 3).

44 Table 2. Isolated yields, HIV-1 PR enzyme inhibition data and antiviral activity of compounds 23a–f, 24a

Yieldb K EC CC Compound R = i 50 50 (%) (nM) ( M) ( M) 13v - - 2.8 0.17 >10

23a 66 6.2 2.0 >10

(S)-23ac 28 >5000 >10 >10

23b 81 65 >10 >10

23c 18 2.7 2.1 >10

23d 29 18 2.6 >10

23e 76 170 8.2 >10

23f 3 130 >10 >10

24d 71 2700 >10 >10

ATZ168 - - 2.7 0.004 - aConditions: see Scheme 5. Inhibitor 13v included for comparison. bIsolated yields in the final reductive amination deprotection step (Scheme 5, d). c(S)-configuration at the tertiary alcohol. d23a with the tertiary alcohol TBS-protected.

45 Table 3. Isolated yields, HIV-1 PR enzyme inhibition data and antiviral activity of compounds 25a–ja

Yieldb K EC CC Compound R = i 50 50 (%) (nM) ( M) ( M) 13v - - 2.8 0.17 >50

25ac 44 4.6 1.0 >50

25bc 82 3.1 1.0 >50

25cc 73 3.5 1.1 >50

25dc 21 3.6 1.0 >50

25ec 35 4.8 1.2 >50

25fd 30 7.0 1.2 >50

25ge 14 3.4 2.1 >50

25hd 29 6.6 8.9 >50

25i 67 6.3 4.9 >50

25j 75 3.5 3.7 >50 aConditions: See Scheme 6 (25a–h) and Scheme 7 (25i–j). bIsolated yield in the cross-coupling/deprotection step. cSuzuki–Miyaura cross-coupling (Method a). dSonogashira coupling (Method c). eSonogashira coupling (Method b).

Inhibitors 23a, 25a–c and 25g were included in permeability (Papp (Caco-2)) and stability studies (CLint) furnishing good results (Table 4). The two substances 23a and ATZ exhibited equally good permeability, while ATZ had slightly better stability. The best permeability was observed for 25a with the phenyl substitution in P1' but at the cost of stability. The most stable compounds were the pyridyl-substituted compounds 25b–c, which unfortunately also suffered from low permeability.

46 Table 4. Permeability and stability of selected inhibitors P (Caco-2) CL Compound app int (× 10-6 cm/s) (µL/min/mg) 23a 4.6 180 25a 21 >300 25b <1.0 63 25c 1.9 20 25g 6.1 >300 ATZ 5.3 90,140a aData from Wempe et al.179

4.3 X-ray evaluation Inhibitors 25a and 25d were co-crystallised with a drug-resistant strain of HIV-1 protease harbouring the mutations Leu63Pro, Val82Thr and Ile84Val, commonly occurring during treatment with, for example, indinavir, lopinavir or atazanavir.179 The compound–inhibitor complexes with 25a and 25d were refined to 2.0 and 1.8 Å, respectively (Figure 22a–c). The interactions were compared with 13d (Paper I, Figure 20) and ATZ. A complicating factor in the comparison of the inhibitor complexes was the fact that compounds 25a and 25d were rotated 180° in relation to compound 13d and ATZ.63,64,180 In the discussion the labels valid for 25a and 25d are used. The overall conformation of 25a and 25d was in good accordance with 13d as well as with ATZ. The main difference observed was that in ATZ the hydroxyl group was positioned symmetrically over the catalytic aspartic acids (Asp25 and 125), while 13d, 25a and 25d all had an asymmetric positioning due to their longer central motif. The asymmetric arrangement reduced the interaction with Asp25 and Asp125 and the overall numbers of interactions were fewer in 25a and 25d than in ATZ. Strong hydrogen bonds to the structural water molecule co-ordinating Ile50 and Ile150 in the flaps was observed in the new inhibitors, with bond distances of 2.6–3.0 Å. The ability of the protease to accommodate the various natural sub- strates66,74,181 also allows it to adapt to different inhibitor structures.182 This was well exemplified by the changes in the S2–S3 pocket to compensate for the bulky tert-butyl (25d) and the flat indanol amide (13d). The methyl amide in 25d displaced Arg8 by 2.0 Å compared to the complex with 13d, disrupting the edge-on cation– interaction183 (3.6 Å) with the phenyl P1 moiety (Figure 22d). The longer central motif together with the extended P1' position in 25a and 25d, compared to 13d, positioned the P1' diaryl moiety close enough to form edge-on aromatic interactions with Phe153 at 3.3 and 3.5 Å, respectively.

47 a) b)

c) d)

Figure 22. a–c) Comparison of the overall conformations and binding patterns of the compounds 25a (a, PDB code 2xye), 25d (b, PDB code 2xyf), and ATZ (c, PDB code 3el9). Hydrogen bonds are denoted by dashed green lines. d) The S2–S3 pocket accommodating compound 25d (gold, and corresponding enzyme in yellow) and 13d (grey, and corresponding enzyme in white). e) Comparison of interactions between the inhibitors 25a (green), 25d (yellow), 13d (grey) and ATZ (purple) and the enzymatic residues Phe153 and Pro81.

This interaction was not observed for ATZ or 13d (4.0 and 4.4 Å, respectively). The Phe153 side chain was also observed to move 1.0–1.5 Å to accommodate the P1' group. Compounds 25a and 25d showed improved hydrophobic interactions (within 3.2–3.6 Å) with the Pro81, which constitutes part of both the S1' and S3' pockets (Figure 22e).65

5 -Hydroxy -Lactams as Central Moiety in HIV-1 Protease Inhibitors

In the series of HIV-1 PIs comprising a tertiary alcohol, inhibitors with one,146-148 two169 and three-carbon spacers (Papers I and II) were evaluated. Although the compounds containing a two-carbon spacer rendered the best PR inhibition,169 there was still room for improvement in the ligand–enzyme interactions. None of the co-crystallised inhibitors in these series exhibited strong symmetrical hydrogen bonding (<3.0 Å) to both Asp25 and Asp125 (Papers I and II).146-148,169 In order to further improve their inhibitory capacity, a more rigid -lactam structure was incorporated and the alcohol was relocated one position away from the backbone to form a secondary alcohol. The resulting -hydroxy -lactam core structure was evaluated with regard to both stereochemistry and length of the backbone spacer. Compounds with privileged stereochemistry and with both two- and three- carbon spacers were co-crystallised with HIV-1 PI.

Figure 23. Schematic comparison between 13v (Paper I) and the inhibitors 37a–j and 43a–e (Paper III). The -lactam-based inhibitors were evaluated with varied stereochemistry (stereocentres annotated with *) as well as with two and three- carbon spacers (Paper III).

5.1 Chemistry In order to synthesise the -hydroxy -lactam-based inhibitors and to evaluate all four stereoisomers at the -lactam, a method was developed starting from commercially available (S)-4-hydroxydihydrofuran-2(3H)-one, 26a, or (R)-4-hydroxydihydrofuran-2(3H)-one, 26b. A two-step alkylation proce-

51 dure, inspired by previously reported alkylations,184-186 was applied. Treating the starting materials 26a or 26b with LDA, 1,3-dimethyl-3,4,5,6-tetrahydro- 2(1H)-pyrimidinone (DMPU) and a first alkylating agent at 50 °C followed by at second portion of LDA, then a second alkylation agent at 40 °C, afforded the dialkylated -hydroxy -lactams 27a–d in 2–49 % isolated yields (Scheme 8, Path A or B).

Scheme 8. Dialkylation of lactones 26a and 26b

Reagents and conditions: Path A: (a) DMPU, LDA, allyl bromide, dry THF, addition at 50 °C, stirred at 40 °C 1 h; (b) LDA, benzyl bromide, addition at 40 °C, stirred at 30 °C 1 h, 27a 49% isolated yield, 27c 33% isolated yield. Path B: (c) DMPU, LDA, benzyl bromide, dry THF, addition at 50 °C, stirred at 40 °C 1 h; (d) LDA, allyl bromide, addition at 40 °C, stirred at 30 °C 1 h, 27b 2% isolated yield, 27d 5% isolated yield.

In accordance with reports by Meyers et al.187 and others,185,186,188 the first alkylation occurred trans to the directing 4-hydroxyl group favouring facial alkylation (Scheme 8, intermediates [i–iv]). In the second alkylation, the alkylating agent was once more directed by the 4-hydroxyl group to be inserted trans, forcing the first inserted group to be positioned cis to the 4-hydroxy group (Scheme 8, 27a–d). When a method to convert the absolute configurations of 27a and 27c to 27d and 27b, respectively, was introduced enough of these low-yielding products were collected to continue the synthesis. Dess–Martin oxidation of 27a and 27c followed by reduction of the newly formed ketones using NaBH4 afforded 27d and 27b (Scheme 9), with ratios of 5.7:1 and 5.9:1 for 27d:27a and 27b:27c, respectively.189-191

52 Scheme 9. Preparation of 27d and 27b from 27a and 27c

Reagents and conditions: (a) 27a or 27c, Dess–Martin periodinane, DCM, rt, 1 h; (b) NaBH4, 1% methanol in THF, rt 2 h, 27d+27a (5.7:1) 92% isolated yield, 27b+27c (5.9:1) 85% isolated yield.

The low-yielding dialkylation reactions in which the benzyl group was introduced prior to the allyl (27b and 27d) followed the trend reported by Johnson et al.186 with 4-substituted lactams, although Meyers et al.187 did not report the same phenomenon when using 5-substituted lactams. In the case of Meyers’ 5-substituted compounds, the yield was not dependent on the order of addition,187 and Huang et al. showed that stereoselective alkylation was mainly dependent on stereo-electronic factors.192 The dialkylated lactones 27a–d were converted to lactams using TBS- protected indanol amine adopting a methodology developed by Orrling et al.193 Microwave heating of lactones 27a–d in a single-mode applicator at 180 °C for 35 min with the indanol amine in the highly polar ionic liquid 194 1-butyl-3-methyl-imidazolium tetrafluoroborate ([bmim]BF4) gave the corresponding lactams. The alcohol moiety was then protected using TBSOTf under basic conditions to give 29a–d (Scheme 10).195,196

Scheme 10. Lactamisation of lactones 27a–d

Reagents and conditions: i. 27a–d, 28, [bmim]BF4, 180 °C, 35 min. ii. triethylamine, TBSOTf, DCM, 0–25 °C, overnight, giving isolated yields of 29a in 64%, 29b in 53%, 29c in 72% and 29d in 50%.

The prime side moiety 11d was previously synthesised as described in Scheme 3 (Papers I and II), but the method was low-yielding, mainly due to troublesome purification. The low-yielding protocol, together with the use of

53 the highly toxic and environmentally hazardous hydrazine hydrate, moti- vated the development of a new, more robust method to better meet the needs of the lactam project. Hydrazone 31 was synthesized starting from the BOC-protected hydrazine 30 in almost quantitative yield, using a previously reported method.197 Compound 31 was benzylated using 4-bromobenzyl bromide (12d) in toluene with the addition of catalytic amounts of a phase-transfer catalyst (tetrabutylammonium hydrogen sulphate) to improve solubility and increase the reaction rate,198,199 rendering 32. Hydrazone 32 was partially deprotected forming 33 during silica flash column chromatography. Deprotection of 32 and 33 formed the hydrochloride salt 34. Amide bond formation with 8 using EDC, HOBt and NMM152 gave the prime side 11d in an isolated overall yield of 61% (Scheme 11).

Scheme 11. Synthesis of the prime side fragment 11d

Reagents and conditions: (a) acetone, MgSO4, acetic acid (cat.), reflux, 1 h, 98% isolated yield; (b) i. KOH, anhydrous toluene, TBAHS, 50 °C, 20 min. ii. 12d, 100 °C, 2 h, 81% isolated yield; (c) 2 M HCl, THF, reflux, 3 h, quantitative yield; (d) EDC, HOBt, NMM, DCM, 0 °C to 25 °C, 15 h, 77% isolated yield (61% isolated yield over four steps). Steps a and c required no purification.

With the prime side in place, the allylic double bond in the non-prime side moieties 29a–d could be oxidatively cleaved to give the corresponding aldehydes, using osmium tetraoxide and sodium periodate.200,201 The aldehyde intermediates [v–viii] were used in the following reductive amination with the prime side fragment 11d, using Na(OAc)3BH as reducing agent. The TBS-protected inhibitors 35a and 35d were isolated prior to the final deprotection, whereupon the lactam-based two-carbon-spacer inhibitors 36a–d were prepared (Scheme 12). It should be noted that the absolute configuration of the lactam carbon in position three changes from (S) in 29a–d, to (R) in the intermediates [v–viii] (Scheme 12) and inhibitors 36a–j (Scheme 13–14) due to changes in the assigned priority according to the sequence rule.150 The three-carbon

54 inhibitors (42a–e) will however, retain the absolute configuration assignment of 29a–d (Figure 24).

Scheme 12. Synthesis of the two-carbon-tethered lactam-based inhibitors 36a–d

Reagents and conditions: (a) OsO4, NaIO4, THF/H2O, rt, overnight; (b) 11d, acetic acid, Na(OAc)3BH, dry THF, rt, overnight, gave 35a in 35% isolated yield and 35d in 54% isolated yield from 29a and 29d, respectively; (c) TBAF, THF, rt, overnight, provided isolated yields of 36a in 38%, 36b in 60%, 36c in 46%, 36d in 34% from 29a–d, respectively.

TBS-protected inhibitors 35a and 35d were used to generate a small series of inhibitors with functionalised P1' moieties. P1' decorations were performed in sealed reaction vessels in a single-mode microwave applicator at 140 °C for 20 min. Palladium-catalysed Suzuki–Miyaura cross-coupling with 3- and 4-pyridylboronic acid, Herrmann’s palladacycle as the palladium precata- 158 160 lyst, [(t-Bu)3PH]BF4 and potassium carbonate as base, rendered inhibitors 36e-h in good isolated yield (Scheme 13).

Figure 24. The nomenclature rules lead to a change in the name of the assigned absolute configuration since the oxygen in the aldehyde intermediates and the nitrogen in the hydrazides (36a–d) have higher priority than the allyl carbon in 29a–d. The nomenclature is unaltered in inhibitors with a three-carbon spacer.150,151

55 Scheme 13. Functionalisation of the P1' moiety in lactam-based two-carbon-tethered inhibitors 36e–h

Reagents and conditions: (a) i. 35a or 35d, Herrmann’s palladacycle, K2CO3, 2- or 3-pyridylboronic acid, [(t-Bu)3PH]BF4, DME, water, 140 °C, 20 min. ii. TBAF, THF, rt, overnight, providing isolated yields of 13g in 63%, 13h in 59%, 13i in 74% and 13j in 66% (Table 6).

To be able to use the 2-pyridyl as part of the P1' moiety a different synthetic route was chosen. Initially, the alcohols 37a and 37d, isolated as side products in the reductive amination to produce 35a and 35d, respectively, were transformed into the corresponding aldehydes via Dess–Martin periodinane- mediated oxidation. Reductive amination introduced the prime side moiety 11h, synthesized as described in above (Section 3.1, Scheme 3), to give inhibitors 36i–j (Scheme 14).

Scheme 14. Synthesis of the 2-pyridyl-functionalised two-carbon-tethered inhibitors 36i–j

TBSO O TBSO O O N N H OH a O HN N O b N H O TBSO TBSO

37a (3R,4S) [v (3R,4S)] 11h 37d (3R,4R) [viii (3R,4R)]

N N

TBSO O O HO O O N H c N H N N O N N O N N H H O O TBSO HO 38a (3R,4S) 36i (3R,4S) 38d (3R,4R) 36j (3R,4R) Reagents and conditions (a) Dess–Martin periodinane, dry DCM, rt, 1 h; (b) acetic acid, Na(OAc)3BH, dry THF, rt, overnight; (c) TBAF, THF, rt, overnight, provided isolated yields of 36i in 63% and 36j in 38% isolated yield from 37a and 37d, respectively.

56 To evaluate the effects of both two- and three-carbon spacers in the lactam- based inhibitors, three-carbon-tethered inhibitors were also synthesized starting from the allylic compound 29a. Only the best stereoisomer from the series with two-carbon spacers was evaluated with an elongated backbone. Compound 29a was refluxed with 9-borabicyclo[3.3.1]nonane (9-bbn) in THF, followed by treatment with hydrogen peroxide under basic conditions to generate alcohol 39.202 Subsequent Parikh–Doering oxidation203,204 using 50% sulphur trioxide pyridine (SO3Py) in dimethyl sulphoxide and triethyl amine gave aldehyde 40 (Scheme 15).

Scheme 15. Synthesis of inhibitors 42a–e with a three-carbon tether

Reagents and conditions (a) i. 9-bbn, dry THF, 80 °C, 6 h. ii. 2 M NaOH, 30% H2O2 in H2O, ethanol, rt, 2 h, 78% isolated yield; (b) Et3N, 50% SO3Py in DMSO, dry DCM, 0–20 °C, 3 h; (c) 11d, acetic acid, Na(OAc)3BH, dry THF, 35 °C, 3 h, 35% isolated yield; (d) TBAF, THF, rt, overnight, giving 42a in 61% isolated yield; (e) i. Herrmann’s palladacycle, K2CO3, arylboronic acid, [(t-Bu)3PH]BF4, 105 °C, 1.5 h. ii. TBAF, THF, rt, overnight, giving 42b in 45% isolated yield, 42c in 35% isolated yield and 42d in 30% isolated yield; (f) i. (tributylstannyl)- 2-pyridine, Pd(PPh3)2Cl2, CuO, DMF, 105 °C, 2 h. ii. TBAF, THF, rt, overnight, giving 42e in 16% isolated yield.

Aldehyde 40 was used in reductive amination with 11d, following the same protocol as with the two-carbon-tethered inhibitors using Na(OAc)3BH, to form the TBS-protected inhibitor 41. Compound 41 was either deprotected

57 to give inhibitor 42a or functionalised using Suzuki–Miyaura or Stille cross- coupling, which after deprotection gave inhibitors 42b–e (Scheme 15, Table 7). The procedure for the Suzuki–Miyaura cross-coupling to give 42b–d followed that used for compounds 36e–h. However, the temperature was reduced to 105 °C and the time extended to 1.5 h. Stille cross-coupling introduced the 2-pyridyl functionality in the P1' position. The 2-(tributylstannyl)- pyridine was coupled with 41 using bis(triphenylphosphine)palladium chloride as the palladium catalyst together with copper oxide.205,206 The reaction mixture was heated to 105 °C for 2 h in a sealed reactor in a single- mode microwave applicator. TBAF-mediated deprotection generated inhibitor 42e in 16 % isolated yield (Scheme 15, Table 7).

5.2 Biological evaluation To enable comparison with previous series of inhibitors (Papers I and II) indanol amide was used as the P2–P3 moiety and the hydrazide-containing prime side as the P1'–P3' moiety. All four stereoisomers of the lactam core structure with a two-carbon backbone tether were evaluated in enzyme- and in cell-based assays (Table 5). The stereochemistry present in the compounds with the best biological activity from the first part of the investigation was used in P1'-functionalised inhibitors (Table 6). The favoured stereochemistry (3R,4S) from this second part was used in the inhibitors synthesized to evaluate the effect of a three-carbon spacer (Table 7). In the first part of the evaluation of the lactam-based inhibitors, the two epimers (3R,4S) (36a) and (3R,4R) (36d) were found to be the most potent compounds, with Ki values <7 nM and EC50 values <1 µM. The biological activity was more dependent on the orientation of the phenyl group in the 3- position than on the orientation of the 4-hydroxy group. However, blocking of the 4-hydroxy group (35a and 35d) led to inactive compounds (Table 5). Functionalisation of the P1' para-positions with 2-, 3- and 4-pyridyls gave up to more than 10-fold improvement in activity according to the cell-based assay (Table 6, 36e and 36g). These lactam-based inhibitors with central two-carbon spacers provided more potent inhibitors than previously seen with linear three-carbon-tethered inhibitors (Papers I and II). However, they did not exhibit the high potency observed with the linear inhibitors with two- carbon spacers reported by Mahalingam et al.169 Elongation of the lactam-based inhibitors to comprise a three-carbon backbone spacer was not beneficial. The elongated inhibitors were at best about 17 times less potent than the two-carbon series (36g vs. 42c). Compared to linear three-carbon inhibitors (Paper I) the lactam inhibitors were at best equipotent in the cell-based assay (e.g. 13t compared to 42b). In

58 the series with three-carbon-tethered inhibitors cytotoxicity was commonly observed, with CC50 values between 5.9 and 31 µM.

Table 5. HIV-1 PR enzyme inhibition, antiviral activity in cell-based assay and cytotoxicity of inhibitors 35a, 35d and 36a–d Yielda K EC CC Compound Structure i 50 50 (%) (nM) (µM) (µM)

35a 35 760 >10 >50

35d 54 800 >10 >50

36a 38 2.1 0.64 >50

36b 62 1200 >10 15

HO O O N H 36c N N O 46 940 >10 >50 N H O HO

36d 34 6.4 0.35 >50

aIsolated yields following the final reductive amination (35a, 35d) or reductive amination deprotection step (36a–d).

59 Table 6. HIV-1 PR enzyme inhibition, antiviral activity in the cell-based assay and cytotoxicicy of inhibitors 36e–j Yielda K EC CC Compound Structure i 50 50 (%) (nM) (µM) (µM)

36e 63 0.8 0.040 >50

36f 59 0.7 0.10 >50

36g 74 0.8 0.040 >50

36h 66 1.7 0.096 >50

36i 48 1.7 0.19 28

36j 38 2.0 0.18 >50

aIsolated yields in the coupling/deprotection step for 36e–h, or from 37a or 37d for 36i—j.

60 Table 7. HIV-1 PR enzyme inhibition, antiviral activity in the cell-based assay and cytotoxicicy of inhibitors 42a–e Yielda K EC CC Compound Structure i 50 50 (%) (nM) (µM) (µM)

42a 61 13 3.9 26

HO O 42b N O 45 12 0.85 15 H N N O N H HO O

HO O 42c N O 35 5.3 1.8 23 H N N O N H HO O

42d 30 4.2 0.7 5.9

HO O 42e N O 16 6.3 2.5 31 H N N O N H HO O aIsolated yields following the deprotection step for 42a, isolated yields in the coupling/deprotection step for 42b–e.

The permeability and stability of selected inhibitors were evaluated, and the results are summarised in Table 8, where ATZ is also included for comparison. Compound 42a was observed to have high permeability (>20 × 10-6 cm/s) but unfortunately the intrinsic clearance was also high. All other -6 inhibitors studied showed intermediate permeability (20 × 10 cm/s > Papp >3 × 10-6 cm/s). When the inhibitors were functionalised with a heteroaromatic P1' moiety the stability increased (Table 8, 36e, 36f, 42c and 42e).

61 Lactam-based inhibitors with permeability and stability properties similar to those of ATZ were identified.

Table 8. Permeability and stability of selected inhibitors P (Caco-2) CL Compound app int (× 10-6 cm/s) (µL/min/mg) 36a n.d. >300 36d 17 >300 36e 3.8 230 36f 5.1 120 42a 22 >300 42c 4.4 122 42e 3.4 130 ATZ 5.3 90, 140a n.d. = not determined. aData from Wempe et al.179

5.3 X-ray evaluation Three inhibitors (36i, 42b and 42d) were co-crystallised with a drug-resistant strain of HIV-1 PR (Leu63Pro, Val82Thr, Ile84Val).74 Since inhibitors 42b and 42d displayed similar binding modes, only 42d was studied as its complex could be interpreted at higher resolution. Compound 36i, with the two-carbon tether, had one more direct hydrogen bond to the enzyme than the three-carbon-tethered inhibitor 42d. The introduction of the novel lactam moiety was well accepted and fitted into the binding site without affecting the overall binding pattern compared to the previously discussed structures; 13d, 18 (Figure 20) and 25a, 25d (Figure 22a–c).

N N

HO O O H HO O O N N O O O OH O N N O H H H H N N N O N N N O N N N O H N H OH N O N N O H OH HO HO O H O H O H O 36i 42d 13d ATZ Figure 25. a–b) Comparison of the overall conformations and binding patterns of compounds 36i (a, PDB code 4a4q, gold) and 42d (b, PDB code 4a6c, purple) in the active site of HIV-1 protease. Hydrogen bonds are denoted by dashed green lines. c) Superimposition of 36i and 42d with interactions with the catalytic aspartic site. d–f) Comparison of the positioning of the co-crystallized inhibitors in the S2–S3 pocket and interaction with Pro81 and Phe153 in S1' pocket. The effect on the S2– S3 site is visualized at residues Asp29, Asp30, Arg108, and Pro181. d) Superimposi- tion of 36i and 42d. e) Superimposition of 42d and 13d (blue). f) Superimposition of 36i and ATZ (black).

62 a) b)

c) d)

e) f)

63 As can be seen from Figure 25c, the loss of interaction between 42d and Asp125 could be explained by the different spatial conformation of the lactam moiety as a result of the longer central backbone. The difference in positioning of the -hydroxy oxygen in the lactam moiety in 36i and 42d was 2.1 Å. Despite this difference, the position of the indanol amide was only affected to small extent. Even when compared with the linear inhibitor 13d (Figure 25e), the variation in the P2–P3 group was limited. The interactions at the S1' site, reported in Paper II, were also observed in the lactam-containing inhibitors. Both 36i and 42d have hydrophobic interactions with Pro81 (3.3–3.8 Å) and edge-face – interactions with Phe153 (3.7–3.8 Å). The Phe153 and Pro81 moieties adopted well to accommodate the P1' variations and to maintain the van der Waals interactions and to prevent clashes with the inhibitors (Figure 25d–e). The amino acid side chains of Phe153 and Pro81 moved 0.9–2.5 Å and 0.5–1.7 Å, respectively, to maintain the beneficial interactions. These interactions were not observed with inhibitor 13d (Figure 25e) or ATZ (Figure 25f).

64 6 Non-Resonant Applicator for Microwave- Assisted Organic Synthesis in a Continuous Flow System

As described in Section 1.2.1, the introduction of microwaves irradiation as an energy source started a new paradigm in the heating of chemical reactions. However, it is associated with some disadvantages: the scale is limited, especially when using single-mode applicators, and the hetero- geneous field commonly observed in multimode applicators makes accurate temperature measurements difficult. The benefits of fast, efficient heating decrease when the scale is increased, resulting in longer heating and cooling times.117 To circumvent some of these problems, microwave heating has recently been combined with flow systems in continuous-flow133-135 and stop/flow approaches.133,207 The implementation of a novel CF-MAOS instrument with a non-resonant microwave applicator, designed specifically for CF applications, allowed efficient heating of high- and low-microwave-absorbing solvents, as well as fast optimisation of reaction parameters and the isolation of products from model reactions. The first attempt to conduct a scale-out process was also investigated.

6.1 The CF-MAOS Instrument The primary components of the prototype CF-MAOS instrument are a microwave generator, an applicator (to transfer energy to the reaction), a tubular reactor (made of a microwave-transparent material such as borosilicate glass) and a control unit (software). Both the power (0–150 W) and the frequency (2.40–2.50 GHz) of the microwave generator can be regulated by the software, which receives input from one of the five IR temperature sensors mounted on the applicator. The generated microwaves are transmitted through a non-magnetic, high- temperature-shielded 50 solid-core coaxial cable to the applicator. Non- absorbed microwaves are reflected back through the coaxial cable and deflected to a dummy load cooled by active coolant circulation.

65 Stock solution 1

1 Mixer Antenna 2 IR sensors

3 Stock solution 2 4

Microwave generator Tubular reactor

Back- Product pressure mixture regulator Figure 26. Schematic overview of the CF-MAOS instrument. The IR sensors are positioned 31.5, 52, 75, 97 and 119 mm from the top of the applicator cavity.

The axial-field microwave applicator is based on a novel design using a non- resonant structure that suppresses mode patterns (standing waves) in the applicator, thus avoiding hot and cold spots. As opposed to single- and multimode applicators, the axial field applicator is not based on the cavity resonator principle. In the axial field applicator the microwave field is generated in a coil surrounding the flow reactor, allowing the microwave field to be concentrated axially inside the coil. The coil is automatically tuned to maximise the heating in the reactor tube by changing the frequency. Reactors of different sizes can be used by changing the length and diameter of the coil in the applicator, allowing the optimisation of reaction conditions such as residence time and flow capacity.208 The applicator is made of a non- magnetic material that contains the microwave field. The applicator used in this project was equipped with five Optris CT IR sensors, allowing the temperature variation to be measured along the length of the reactor. The 200-mm-long tubular reactor is inserted inside the 90 mm long antenna, which heats approximately 100 mm of the reactor (up to 150 mm depending on the dielectric properties of the reactor content). The reason heating occurs outside the 90 mm antenna is the electric component of the electromagnetic field, which radiates in the transverse direction all along the heating zone, propagating towards the outlet of the reactor. The reactors used were made of microwave-transparent borosilicate glass, and had an inner lumen of 3 mm and a maximum outer diameter of 9 mm. The reactors have steel connectors with Valco® fittings and can easily be removed for washing or replacement when necessary. In these first studies, it was assumed in the residence time calculations that the heated zone was 100 mm, corresponding to a volume of 0.7 mL. The software controls both the output power and frequency of the microwaves, which can automatically be adjusted based on signals from one of the five IR temperature sensors. The user can also set a specific temperature or a

66 fixed microwave output power. A two-channel syringe pump with a pressur- ised storage compartment, a static mixing chamber and a 17-bar back- pressure regulator are also included in the set-up.

Figure 27. Borosilicate glass reactors designed for the CF-MAOS instrument. Length 200 mm, inner 3 mm (left). The reactors are connected to the system using standard Valco® connectors (right).

Figure 28. The applicator (foreground) connected to the MW generator (red box in the background) via a coaxial cable. The inlet to the reactor is visible on the top of the applicator.

Figure 29. IR camera image of the applicator during heating of an oxidative Heck reaction mixture to 200 °C. Slightly lower temperatures were observed at the inlet of ambient tempered reaction mixture at the top. The temperature was also observed to decrease when the mixture left the MW field.

67 6.2 Evaluation of Instrument Performance Initial experiments were designed to test the capability and robustness of the instrument. Common MOAS solvents were heated from room temperature to temperatures 20 °C below the normal boiling point (bp), to the bp and to 20 °C above the bp to determine heating profiles and heating ramp times. A flow of 701 µL/min, corresponding to a residence time of 1 min, was used. The temperature was easily kept within the set temperature ±2 °C using manual or automatic temperature control. In the initial experiments, the temperature was monitored using sensor 2 since it showed the fastest response (Figure 30). Heating rates were then measured from room temperature to the bp of each solvent using the standard 3mm reactor with a residence time of 1 min. The heating rates were in accordance with the tan of the solvent, as expected (Figure 31). Despite the relatively low power of the microwave generator compared with most batch applicators (max 150 W vs. 300- 1500 W209,210), the output was still sufficient to heat the solvents rapidly to the target temperatures. Since tan is temperature dependent (decreasing with increasing temperature, Figure 16) the possibility of adjusting the microwave frequency while heating partly compensates for this change, allowing the energy efficiency to be optimised.

250 Set Temp, bp -20 °C Average 1-4, bp -20 °C Set Temp, bp Average 1-4, bp 200 Set Temp, bp +20 °C Average 1-4, bp +20 °C

150

Temperature100 (°C)

Figure 30. Results of temperature stability measurements. Solvents commonly used in MAOS were heated to bp 20 °C, to bp and to bp +20 °C. ‘Average 1–4’ is the average temperature observed for sensors 1–4 positioned within the heated zone.

68 6 1

0.9

5 0.8

0.7 4

0.6

0.5 3 tan

0.4

2 Heating rate (°C/s) 0.3

0.2 1

Heating rate 0.1 tan d 0 0

Figure 31. Results of heating rate measurements on model solvents. For acetone, DMF, NMP and water, heating was software controlled. For acetonitrile, dioxane, DMSO and ethanol, heating was manually controlled. The heating rate was measured from 25 °C to the solvent bp. No tan data were available for 1,4-dioxane.

To verify the uniformity of the heating profile over the length of the reactor observed via the five IR sensors, one of the walls of the applicator was removed and images were obtained using an FLIR® IR camera. From these images it could be concluded that the temperature of the antenna itself did not increase to the same extent as the reaction mixture, although the reaction mixture was efficiently heated, and that the reaction mixture was evenly heated within the antenna. However, there was a tendency for a warmer region to form around the fourth turn of the antenna. As expected, and observed from the IR sensors, the temperature decreased outside the microwave field (Figure 29). Safety measurements were performed outside the applicator during all microwave heating experiments and chemical reactions to detect microwave irradiation. No irradiation was detected on any occasion. Furthermore, no microwave irradiation was measured outside the applicator cavity, even when the applicator was opened for the recording of IR camera images.

69 6.3 Evaluation of Model Reactions Initial evaluation of chemical reactions using the novel non-resonant CF- MAOS system focused on investigating the instrument’s ability to rapidly screen reaction conditions (by changing the temperature and residence time), and determining the chemical throughput. Proof-of-principle studies were set up employing a range of solvents (low- and high-microwave-absorbing) temperatures and flow regimes, as well as metal-catalysed reactions.

Pd(II)-catalysed oxidative Heck vinylation of an arylboronic acid The first report of palladium(II)-catalysed oxidative Heck vinylation of arylboronic acids was presented by Lindh et al. in 2009.211 These conditions were optimised a year later and applied in a conductively heated CF set-up by Odell et al.128 The cross-coupling between 4-biphenylboronic acid and vinyl acetate to form 4-vinyl-1,1'-biphenyl was chosen as a model reaction (Scheme 16). In the original protocol the reaction mixture was heated in microwave batch conditions to 140 °C for 30 min using a single-mode applicator.211 When the protocol was adapted for CF conditions using conductive heating, Odell et al. choose 150 °C and a 2 min residence time as the preferred reaction conditions.128 These optimised flow conditions provided an excellent starting point when adapting the reaction to the CF-MAOS system.

Scheme 16. Pd(II)-catalysed oxidative Heck vinylation of 4-biphenylboronic acid

Reaction conditions: Pd(OAc)2, 1,3-bis(diphenylphosphino)propane, DMF, 140 °C, 75 s residence time, gave 45 in 66% isolated yield, calculated throughput 2.83 mmol/h (0.51 g/h).

Initial precipitation problems when using 0.5 M stock solutions were avoided by reducing the concentration in the stock solutions to 0.25 M. The temperature and residence time were then varied between 120 and 160 °C and 30 and 120 s, respectively. Nine combinations of operating conditions were evaluated (Table 9). Using the internal standard 4-methoxybenzonitrile the preferred conditions were chosen from the product/internal standard (IS) ratio as determined by GC/MS. The temperature could be reduced slightly, but more importantly, the residence time could be reduced from 120 s to 75 s with a retained isolated yield of 66% (compared to the 68% isolated yield reported by Odell et al.).128

70 Table 9. Optimisation of reaction conditions for Pd(II)-catalysed oxidative Heck vinylation of a 4-biphenylboronic acid Run Residence time Set temperature Flow/pump Product/IS (s) (°C) (µL/min) 1 120 120 177 1.11 2 120 140 177 1.09 3 120 160 177 1.22 4 75 120 283 1.25 5 75 140 283 1.42 6 75 160 283 1.11 7 30 120 707 0.98 8 30 140 707 1.19 9 30 160 707 1.28 10a 75 140 283 aFlow process for isolated yield, giving 66%.

Suzuki–Miyaura cross-coupling Another metal-catalysed reaction was selected as the next model reaction. Since it was first reported as a palladium(0)-catalysed cross-coupling reaction between an organoboron compound and an organic halide154,155 the Suzuki–Miyaura cross-coupling reaction has become commonly used by both medicinal, organic and process chemists. The Suzuki–Miyaura type of reaction is well suited for use under MAOS conditions,99,156 and is frequently reported under microwave batch conditions and CF conditions. However, there are few reports on its application in CF-MAOS, although examples have been previously published by, e.g., Wilson et al.,136 Baxendale et al.138 and Comer and Organ.141 To investigate the possibility of running Suzuki–Miyaura reactions in the new non-resonant instrument the cross-coupling between 4-bromobenzyl methyl ether and 4-methoxyphyenylboronic acid was evaluated (Scheme 17).

Scheme 17. CF-MAOS Suzuki–Miyaura cross-coupling

Reaction conditions: DBU, Pd(PPh3)2Cl2 in DMF/water, 150 °C, 30 s residence time, giving 48 in 71% isolated yield, with a calculated throughput 3.0 mmol/h (0.69 g/h).

Two stock solutions were prepared, one containing the palladium pre-catalyst (Pd(PPh3)2Cl2) in DMF (0.005 M), and the other containing the arylbromide, the arylboronic acid and DBU as a base212 dissolved in DMF/water (0.1 M). The temperature and residence time were varied between 130 and 160 °C and 10 and 150 s, respectively, (Table 10). The

71 preferred conditions (Run 11) gave 71% isolated yield with a calculated throughput of 3.0 mmol/h (0.69 g/h).

Table 10. Optimisation of reaction conditions for Suzuki–Miyaura cross-coupling Run Residence time Set temperature Flow/pump Conversion (min) (°C) (µL/min) (%) 1 2.50 140 141 80 2 2.50 150 141 Full 3 1.00 160 353 Full 4 0.50 150 707 Full 5 1.50 150 236 Full 6 2.75 150 129 Full 7 1.75 150 202 Full 8 0.17 150 2121 81 9 0.17 140 2121 47 10 0.17 130 2121 21 11a 0.50 150 707 Full aFlow process for isolated yield, giving 71%.

Oxathiazolone synthesis To evaluate the use of the CF-MAOS instrument in a drug discovery project, attention was turned towards 1,3,4-oxathiazol-2-ones, which have recently been used as selective M. Tuberculosis (Mtb) proteasome inhibitors.213,214 The 1,3,4-oxathiazol-2-ones are common C=N-S synthons (especially in heterocycles),215-218 and have also been used in fungicides.219,220 To synthesise the 5-phenyl-1,3,4-oxathiazol-2-one a 0.25 M stock solution of benzyl amide and a 0.75 M stock solution of chlorocarbonyl- sulphenyl chloride were prepared, both in 1,4-dioxane (Scheme 18). The stock solutions were pumped at identical flow rates through the reactor while applying temperatures ranging from 120 to 220 °C, with residence times varying from 40 s to 5 min (Table 11). The preferred conditions (200 °C, 1 min) gave the product in 62% isolated yield and with a calculated throughput of 3.3 mmol/h (0.58 g/h).

Scheme 18. CF-MAOS oxathiazolone synthesis

Reaction conditions: 1,4-dioxane, 200 °C, 1 min residence time, gave 51 in 62% isolated yield, with a calculated throughput 3.3 mmol/h (0.58 g/h).

72 Table 11. Optimisation of reaction conditions for oxathiazolone synthesis Run Residence time Set temperature Flow/pump Conversion (min) (°C) (µL/min) (%) 1 5.00 120 71 96 2 2.50 130 141 95 3 1.25 130 283 91 4 1.25 140 283 91 5 1.25 150 283 92 6 1.25 160 283 94 7 0.67 160 530 88 8 0.67 170 530 90 9 1.25 180 283 96 10 1.25 220 283 98 11 1.25 200 283 99 12a 1.00 200 353 Full aFlow process for isolated yield, giving 62%.

Fischer indole synthesis The indole moiety has proven its usefulness as part of a variety of biologi- cally active compounds.221 A convenient method of synthesising indole- containing compounds is Fischer indole synthesis.222 The use of Fischer indole synthesis has been reported in a microwave batch protocol,223 in a micro-CF protocol224 and even in CF-MAOS protocols.139 Scale-out in a CF system with traditional heating has also been reported.225 Two stock solutions were prepared; one containing a 1.0 M solution of phenylhydrazine and one with 1.1 M cyclohexanone, both in a 3:1 acetic acid/2-propanol solvent (Scheme 19).

Scheme 19. CF-MAOS Fischer indole synthesis

Reaction conditions: acetic acid/2-propanol (3:1), 230 °C, 20 s residence time, gave 54 in 90% isolated yield, with a calculated throughput 57.2 mmol/h (9.8 g/h).

Reaction conditions with temperatures between 210 and 230 °C and with residence times between 0.33 and 5 min were evaluated (Table 12). Although the highest conversion was observed at 210 °C and a residence time of 2.5 min (Table 12, Run 2), the preferred conditions were 230 °C with 0.33 min residence time (Table 12, Run 8) due to the higher throughput achieved with the short residence time (62% isolated yield, calculated throughput 57.2 mmol/h (9.8 g/h)).

73 Table 12. Optimisation of reaction conditions for Fischer indole synthesis Run Residence time Set temperature Flow/pump Conversion (min) (°C) (µL/min) (%) 1 5.00 220 142 97.4 2 2.50 210 284 99.7 3 2.50 220 284 99.6 4 1.25 220 568 99.4 5 1.00 230 706 98.3 6 0.63 230 1136 97.7 7 0.33 230 2120 98.0 8a 0.33 230 2120 98.9 aRun for isolated yield, giving 90%.

Claisen rearrangement Claisen rearrangement was among the first reactions studied with microwave heating.101 The high temperature and long reaction times normally needed make the reaction well suited for MAOS,226-228 but the question was whether reaction times could be reduced sufficiently to make the reaction suitable for CF-MAOS. CF synthesis using classical heating has been reported, for example, by Razzaq et al.225 and Kong et al.229 As model substrate 4-allyloxianisole was chosen since it has been concluded that electron-withdrawing groups in the para-position increase the reaction rate.230 As a medium with a high bp was required, NMP was used for preparation of the 2.0 M stock solution (Scheme 20).

Scheme 20. CF-MAOS Claisen rearrangement

Reaction conditions: a) NMP, 270 °C, 5 min residence time, gave 56 in 79% isolated yield, calculated throughput 13.6 mmol/h (2.23 g/h). b) neat, gave 56 in 85% isolated yield, calculated throughput 15.0 mmol/h (2.46 g/h).

The reaction conditions were optimised by evaluating temperatures between 225 and 270 °C and residence times of 1–5 min (Table 13, Runs 1–12). The preferred reaction conditions (Table 12, Run 12) gave 79% isolated yield and a calculated throughput of 13.6 mmol/h (2.23 g/h). Inspired by these encouraging results, Claisen rearrangement was also attempted under neat conditions (Table 12, Runs 13–20). Longer residence times were required under neat conditions. The preferred conditions

74 (Table 12, Run 20) gave an isolated yield of 85% and an impressive calculated throughput of 15.0 mmol/h (2.46 g/h).

Table 13. Optimisation of reaction conditions for Claisen rearrangement Run Residence time Set temperature Flow Conversion (min) (°C) (µL/min) (%) 1 5.0 225 71 96 2 3.0 225 141 95 3 1.0 240 283 91 4 3.0 240 283 91 5 5.0 240 283 92 6 5.0 250 283 94 7 3.0 250 530 88 8 3.0 260 530 90 9 1.0 260 283 96 10 5.0 260 283 98 11 5.0 270 283 99 12a 5.0 270 353 Full 13 5.0 250 141 53 14 5.0 230 141 36 15 5.0 270 141 75 16 10.0 250 71 77 17 10.0 230 71 56 18 15.0 250 71 85 19 15.0 270 47 95 20b 15.0 260 47 96 aFlow process for isolated yield, giving 79%. bFlow process for isolated yield, giving 85%.

Diels–Alder reaction The Diels–Alder reaction is commonly used as a model reaction in MAOS, often requiring long reactions times (10–60 min) and temperatures of 160– 180 °C.231-233 In a recent CF-protocol, using 2,3-dimethyl-butadi-1,3-en and propenenitrile, a 2 min residence time was reported, but at an elevated temperature. The temperature was set to 280 °C, but recorded temperature was up to approximately 330 °C.130 In a first model reaction isoprene and maleic anhydride was evaluated as reagents. In acetonitrile full conversion was achieved at 80 °C with a 1 min residence time. However, it was soon observed that the reaction occurred spontaneously at room temperature, showing that it was not useful as a model transformation. A change to the less reactive 1,4-naphthoquinone, reported in Diels–Alder reactions as early as 1942,234 allowed further optimisation. In the first experiments using 1,4-naphthoquinone and isoprene in this work, acetonitrile was chosen as the solvent, but only 73% conversion was observed at 190 °C. Using acetonitrile and 17 bar back pressure, the reaction temperature could not be increased above 190 °C, without encounter

75 problems with boiling. Changing to NMP with a higher boiling point made an increase in temperature possible. However, the side products increased at the higher temperature. Running the reaction in NMP at 190 °C with a 5-minute residence time gave >95% conversion and an isolated yield of 52%, with a calculated throughput of 4.72 mmol/h (1.07 g/h), Scheme 21.

Scheme 21. CF-MAOS Diels–Alder reaction

Reaction conditions: NMP, 5 min residence time, 190 °C giving 59 in 52% isolated yield and with a calculated throughput of 4.72 mmol/h (1.07 g/h).

The non-resonance applicator provided the possibility to rapid optimisation and access to product in the model reactions evaluated. Temperatures between 140 and 270 °C and residence times between 20 s and 15 min were applied and both high- and low-microwave absorbing solvents were used.

76 7 Concluding Remarks

The work presented in this thesis contributes to the understanding of drug- like structures inhibiting the HIV-1 protease. Two new scaffolds have been presented together with biological evaluation using enzyme- and cell-based assays. X-ray analyses of co-crystallised inhibitors revealed binding modes and inhibitor–enzyme interactions. Furthermore, the use of a novel non- resonant continuous-flow microwave instrument allowed rapid online optimisation of reaction parameters directly applicable for the isolation of the product.

Protease inhibitors The backbone tether in previously presented inhibitors containing tertiary- alcohols was elongated to a three-carbon spacer using indanol amide as the P2–P3 moiety and a hydrazide containing prime side part. The P1' position was functionalised, and the best PIs obtained exhibited values of Ki = 2.3 nM and EC50 = 0.17 µM. Inhibitors with high permeability in the Caco-2 assay were identified.

Thereafter, P2–P3 alternatives to the indanol amide were evaluated. The use of tert-butyl methyl amide as P2–P3 moiety provided inhibitors with potency similar to the first series, but without the metabolically unstable indanol moiety, and previously unreported interactions with the Phe153 were identified.

Novel -hydroxy -lactam-based inhibitors were evaluated with regard to stereochemistry and backbone length (two- or three-carbon tethers). A small series of variations in the P1' position was also included. The novel scaffold of enzyme–inhibitor complexes with the inhibitors exhibiting the stereochemistry that gave the most potent inhibitors was thoroughly evaluated by X-ray analysis. The best inhibitor in the series exhibited values of Ki = 0.8 nM and EC50 = 0.04 µM.

Finally, a novel non-resonant CF-MAOS instrument was evaluated. The possibility of changing the temperature and residence time rapidly gave the opportunity to optimise reaction conditions, and run experiments for isolated yield within three working hours. The CF-MAOS instrument provided efficient and homogeneous heating, with good control of reaction conditions, including the possibility to perform scale-out processes.

78 Acknowledgments

The work presented in this thesis was conducted at the Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Faculty of Pharmacy, Uppsala University, Sweden, between September 2005 and December 2011. I would like to express my sincere gratitude to the following people, who have been involved in this work over the past six years, and without whom this journey would have been less enjoyable or, in some cases, probably not even possible.

Professor Mats Larhed, my supervisor, for always having time for my constant stream of questions, for all our fruitful discussions, and for just passing by with a few encouraging words when I needed them. Once, back in 1999, I applied to prolong the day to 36 hours, but it was rejected. I guess your application was approved Mats, because that’s the only way I can explain how you always have time to read another draft of a manuscript or poster. Thank you for all your support and immense knowledge.

Professor Anders Karlén, my co-supervisor, for his expertise in modelling and analysis of enzyme–inhibitor complexes, as well as for putting up with all my nagging about teaching schedules.

Professor Anders Hallberg, our Rector Magnificus, and my co-supervisor, who invited us to have the exam in Skåne, and who, as head of the department made it possible for me to start my PhD studies. He started as my supervisor but become my co-supervisor when Uppsala University needed his supervision.

My co-authors: Dr Xiongyu Wu, Dr Jenny K. Ekegren, Dr Advait A. Joshi, Dr Riina K. Arvela and Alejandro Trejos, for their work and effort in the HIV projects; Ashkan Fardost and Dr Francesco Russo, for the flow, Dr Magnus Persson, Dr Johan Unge and Prof. Torsten Unge, for revealing some of the secrets of X-ray crystallography, the team at Medivir AB, including Dr Hans Wallberg, Dr Lotta Vrang, Dr Åsa Rosenquist and Prof. Bertil Samuelsson, for all the biological data, and the team at Wave- Craft AB including Dr Jon-Sverre Schanche and Magnus Fagrell, for giving me the opportunity to take part in the evaluation of the non-resonant microwave applicator.

79 Dr Helen Sheppard, for excellent linguistic revision of my thesis. All linguistic mistakes are my own last-minute changes. Dr Anna Ax, Dr Pernilla Örtqvist, and Marc Stevens for constructive criticism on my thesis.

My roommates and lab-mates: Linda Axelsson for fruitful discussions and for her pertinent questions when reviewing, Dr Riina Arvela for good col- laboration in the lab, Dr Advait Joshi for all the laughs and story-telling, and for at least trying to explain the rules of cricket, and of course for your excellent advices “in the hood”, and Dr Anneli Norqvist for good company while we both were writing. Thanks for hearing me scream on the 5th floor, for your close examination of the figures and schemes, and for alerting me to some of the pitfalls in the process of writing.

My SOFOSKO and Master’s student Sajjad Ahmed, thank you for all your hard work and fun in the lab.

All the staff at the division for making it a pleasant place to work: Gunilla Eriksson for always being supportive and for looking after us PhD students; Eva Strömberg for making arrangements that are realistic and work; Sorin Srbu for making all the bytes work together and for always having a minute for tricky computer-related questions; and Dr Eva Åkerblom and Dr Uno Svensson for all their knowledge and expertise.

Past and present members of the LC team, especially Dr Robert Rönn, for introducing me to the instruments, and Dr Gunnar Lindeberg, our own LC guru who always knows which the right gradient is and who wears pink with such grace.

All past and present members at the division for making it such a great place to work, and for all the fun, especially Rebecca, my “little sister”, who beat me to the finishing line! Anneli, for introducing me to the art of cycling; I never thought I would survive the first trip with you and Alex… My travel- ling companions: Johan and Rebecca, learning different “bag techniques” in Copenhagen. Fredrik, Olle and Kristina for, among other things, finding out that a roof-top pool can be filled with sand and be on floor 3 out of 7, at least in Dubai. How did you always manage to find someone to offer us dinner Kristina? Fredrik, Olle and Hanna, together with our ground support Henrik, for hunting down our missing luggage. Now even a Nobel Prize Laureate know the importance of a very special break... Patrik and Anna-Karin, for their good company at the conference in Boston, and for the fun we had together speed-touristing in N.Y.

80 Anna-Maria Lundin’s Foundation at Smålands Nation and the Swedish Pharmaceutical Society are acknowledged for financial support of my par- ticipation in scientific meetings.

För att ha påmint mig om att det finns en värld även utanför dragskåpen vill jag naturligtvis tacka familj, släkt och vänner. Om någon undrar varför detta tagit så lång tid skulle jag vilja hänvisa till en artikel av D. Upper från 1974 som ger en tydlig bild av problematiken.235

Jag skulle särskilt vilja framföra min stora tack till

De slipsbeprydda herrarna, för många trevliga helger, fula slipsar och illustra tävlingar av många de slag.

Bröder i det bästa av sällskap, för att det var bättre förr.

Anna och Fredrik Ax, för att ni introducerat mig till många spännande och trevliga miljöer och för att jag alltid kan fråga om goda råd.

Christer, Jonny och övriga i Lif för att jag fått fortsätta berika mig i det militära ljuset. Kom ihåg att en bra KVM alltid har allt ni behöver i höger benficka.

Magnus, för alla glada tillrop och för att du trots allt inte gett upp hoppet om mig.

Sonny, för många trevliga luncher och alltid intressanta diskussioner.

Christina och Patrik med familj, för trevliga fikastunder när jag lyckats leta mig västerut.

Ulla och Mats, för många trevliga besök i Linköping, på Hästö och där vi kunnat ansluta medan ni varit på rull.

Lars, för klassiker-utmaningen, när gör vi den igen? För alla roliga stunder i Lysekil och Göteborg.

Mamma och pappa för att ni alltid tror på mig och stöttar mig i allt jag gör.

Lotta, för att du helt enkelt är världens bästa Lotta, för att du står ut med alla mina galna idéer och för att du lärt mig uppskatta ett stort svart lurv med en väldigt söt nos.

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