Design, Synthesis, and Evaluation of Aryl Hydantoins and Ureas As Antischistosomal Agents

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Theses & Dissertations Graduate Studies

Fall 12-16-2016

Design, Synthesis, and Evaluation of Aryl Hydantoins and Ureas as Antischistosomal Agents

Chunkai Wang University of Nebraska Medical Center

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DESIGN, SYNTHESIS, AND EVALUATION OF ARYL HYDANTOINS AND UREAS AS ANTISCHISTOSOMAL AGENTS

Chunkai Wang

Design, synthesis, and evaluation of aryl hydantoins and ureas as antischistosomal agents

Chunkai Wang

A DISSERTATION

Presented to the Faculty of

the University of Nebraska Graduate College

in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy

Pharmaceutical Sciences

Graduate Program

Under the Supervision of Professor Yuxiang Dong

University of Nebraska Medical Center

Omaha, Nebraska

October, 2016

Supervisory Committee:

Yuxiang Dong, Ph.D. Jonathan L. Vennerstrom, Ph.D.

Joseph Vetro, Ph.D. Jialin Zheng, M.D. i

ACKNOWLEDGMENTS

I would like to express my sincere and deep gratitude to my academic advisor Dr.

Yuxiang Dong and the big boss Dr. Jonathan L Vennerstrom for their guidance, mentorship, encouragements and discussions.

I would also like to thank the committee members, Dr. Jialin Zheng and Dr. Joseph

A Vetro, for their valuable scientific inputs.

I am thankful to the members of Dr. Vennerstrom’s research group (Dr. Xiaofang

Wang, Dr. Jianbo Wu, Derek A Leas, Dr. Yogesh A Sonawane, and Dr. Qingjie Zhao) for the support and friendship. I am also thankful to Dr. Sakthivelin Muniyan and Matthew A

Ingersoll in Dr. Ming-Fong Lin’s laboratory for teaching me cell culture. The dissertation could not be achieved without the help of them.

I am thankful to the administrative staff and the faculty of the Department of

Pharmaceutical Sciences at the University of Nebraska Medical Center and the

Department of Chemistry at the University of Nebraska Omaha, especially Katina M

Winters, Elaine Payne, Dr. James Wood, Dr. James Hagen and Dr. Frederic C Laquer, for their suggestions and support during my study.

I would like to thank Edward L Ezell for helping me get started in the NMR Lab. I am also grateful for our collaborator at Swiss Tropical and Public Health Institute, Dr.

Jennifer Keiser.

I wish to express my gratitude to Dr. Gary C Yee and Esther Yee for their immense support and advice during my Ph.D. studies. ii

Last and most importantly, I am thankful to my parents, wife, and two wonderful sons, for their support, sacrifices and patience over the years. iii

Design, synthesis, and evaluation of aryl hydantoins and ureas as

antischistosomal agents

Chunkai Wang, Ph.D.

University of Nebraska Medical Center, 2016

Advisor: Yuxiang Dong, Ph.D.

Schistosomiasis is a tropical parasitic disease caused by infections with flukes of the genus Schistosoma, affecting as many as 440 million individuals worldwide, with 779 million living at risk of infection. A new drug for schistosomiasis is urgently needed as praziquantel (PZQ) is currently the drug of last resort and the development of resistance cannot be ignored, particularly in view of its large-scale use in many endemic countries. Furthermore, PZQ is active against adult but not juvenile schistosomes; this may be an important factor in the frequently observed treatment ‘failures’ in areas endemic for schistosomiasis.

The introduction of PZQ in 1982 likely led to decisions to abandon the development of a number of promising antischistosomal agents that were discovered in the same time period. One of these was AH01, the lead compound from a series of aryl hydantoins that were investigated in some detail at Hoffmann La-Roche. A number of characteristics of AH01 justify it as a compelling antischistosomal prototype. This work confirmed the high antischistosomal efficacy of AH01 and demonstrated that the aryl hydantoin is also effective against juvenile infections. Further, AH01 had no measurable interaction with the androgen receptor in a ligand competition assay, but it did block DHT-induced cell proliferation in androgen-dependent cell line. For this aryl hydantoin, there appears to be no correlation between antischistosomal activity and androgen receptor interaction.

Building on this foundation, a number of AH01 analogs were designed and synthesized to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-AR interactions. This work identified several new derivatives of AH01 iv

with low cytotoxicity and promising in vivo antischistosomal activities that set the stage for further optimization.

This work also discovered that urea carboxylic acid UC00, the product formed by hydrolysis of the hydantoin substructure and AR33, a side-reaction product formed in the synthesis of the aryl hydantoins, have substantial in vivo antischistosomal efficacy. Base on this finding, novel N,N'-diaryl ureas were designed and synthesized as new analogues of AH01. The most promising compound identified in this work was N,N’-di(quinoxalin-2-yl)urea. This new lead compound can serve as a new direction for further optimization.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... i

ABSTRACT...... iii

TABLE OF CONTENTS...... v

LIST OF FIGURES...... viii

LIST OF SCHEMES...... ix

LIST OF TABLES...... x

Chapter 1. Introduction

1.1. Schistosomiasis overview...... 1

1.2. Life cycle of schistosome...... 1

1.3. Global distribution...... 2

1.4. Prevention...... 2

1.5. Clinical features...... 3

1.6. Clinically used drug (Praziquantel) ...... 4

1.7. Aryl hydantoins and antischistosomal activity...... 7

1.8. References...... 11

Chapter 2. Antischistosomal versus Antiandrogenic Properties of Aryl Hydantoin Ro 13- 3978

Abstract...... 19

2.1. Introduction...... 19

2.2. Chemistry...... 22

2.3. Results and Discussion...... 24

2.4. Summary...... 30

2.5. Experimental...... 30

2.6. References...... 37

Chapter 3. Revisiting the SAR of the Antischistosomal Aryl Hydantoin Ro 13-3978 vi

Abstract...... 40

3.1. Introduction...... 42

3.2. Design...... 44

3.3. Chemistry...... 49

3.4. Results and Discussion...... 59

3.5. Summary...... 60

3.6. Experimental Section...... 61

3.7. References...... 92

Chapter 4. Urea carboxylic acid antischistosomal agents

Abstract...... 95

4.1. Introduction...... 95

4.2. Design...... 96

4.3. Chemistry...... 96

4.4. Discussion...... 101

4.5. Summary...... 101

4.6. Experimental...... 105

4.7. References...... 116

Chapter 5. N,N’-diaryl urea antischistosomal agents

5.1. Abstract...... 118

5.2. Introduction...... 118

5.3. Design...... 119

5.4. Chemistry...... 120

5.5. Results and Discussion...... 127

5.6. Summary...... 127

5.7. Experimental...... 131 vii

5.8. References...... 157

Chapter 6. Summary...... 160

APPENDIX: Gas Chromatogram and Mass Spectrogram...... 166

viii

LIST OF FIGURES

Figure 1.1 Antischistosomal agents...... 5

Figure 2.1 Aryl hydantoin structures...... 21

Figure 2.2 AR SPA ligand displacement assay...... 28

Figure 2.3 Effects of aryl hydantoins on DHT-induced LNCaP cell proliferation...29

Figure 2.4 GC spectrum of AH01...... 33

Figure 2.5 MS spectrum of AH01...... 34

Figure 3.1 Principal SAR of AH01...... 41

Figure 3.2 Praziquantel (PZ), aryl hydantoins Ro 13-3978 (1) and nilutamide.....43

Figure 4.1 Proposed mechanism for the formation of UC04...... 102

Figure 4.2 1H NMR of UC05 and UC09 (1:1) ...... 103

Figure 4.3 1H NMR of UC05 and UC09 (20:1) ...... 104

LIST OF SCHEMES

Scheme 2.1 Synthesis of AH01...... 23

Scheme 3.1 Synthesis of AR11, AR62, AR63, AR13, AR18, AR57, AR17 and AR37...... 52

Scheme 3.2 Synthesis of AR59, AR22, AR54, AR43, AR52, AR48 and AR50 ...... 53

Scheme 3.3 Synthesis of AR30, AR16 and AR36...... 54

Scheme 3.4 Synthesis of AR64 and AR28...... 55

Scheme 3.5 Synthesis of AR08, AR53, AR23 and AR35...... 56

Scheme 3.6 Synthesis of AR19b, AR34, AR40, AR29, AR32 and AR51...... 57

Scheme 3.7 Synthesis of AR58 and AR56...... 58

Scheme 4.1 Synthesis of U03 and U06-U08...... 97

Scheme 4.2 Synthesis of U01 and UC04...... 98

Scheme 4.3 Synthesis of UC05...... 99

Scheme 4.4 Synthesis of UC06...... 99

Scheme 4.5 Formation of UC05 and UC09...... 100

Scheme 5.1 Synthesis of AR33, AR42, AR49, U05, U09-U11 and U13-U18...... 122

Scheme 5.2 Synthesis of U07 and U08...... 123

Scheme 5.3 Synthesis of AR41, U04 and AR50...... 123

Scheme 5.4 Synthesis of ARX and AR38...... 124

Scheme 5.5 Synthesis of AR45-AR47...... 125

Scheme 5.6 Synthesis of U03 ...... 126

Scheme 5.7 Synthesis of AR39...... 126

LIST OF TABLES

Table 2.1 Effects of single oral doses of AH01 and AH02 administered to mice harboring 21-day-old juvenile and 49-day-old adult S. mansoni infections...... 27

Table 3.1 Antiandrogenic and antischistosomal data for N1-substituted 4-fluoro-3- trifluoromethylphenyl hydantoins...... 45

Table 3.2 Antiandrogenic and antischistosomal data for N3-substituted aryl

hydantoins...... 46

Table 3.3 Antiandrogenic and antischistosomal data for 5-substituted 4-fluoro-3- trifluoromethylphenyl hydantoins...... 47

Table 3.4. Antiandrogenic, and antischistosomal data for 4-fluoro-3-trifluoromethylphenyl hydantoinheterocyle variants...... 48

Table 5.1 In vitro and in vivo antischistosomal data for urea analogs...... 128

Chapter 1 Introduction

1.1 schistosomiasis overview Human schistosomiasis is the second most common tropical chronic disease caused by genus Schistosoma. There are five species of schistosomes, namely

Schistosoma mansoni, S. japonicum, S. haematobium, S. intercalatum and S. mekongi.

The former three have the widest geographical distributions and are of particular public health and economic impact.1 This disease is a huge burden on the healthcare system in developing countries in the tropics and subtropical regions. There are estimated 600 million people at risk of infection.2 More than 230 million people have been affected by schistosome.3 More than 200,000 deaths annually are caused by a severe schistosomal infection in sub-Saharan Africa. 4

The symptoms of this disease include a rash or itchy skin after becoming infected and fever, chills, cough, and muscle aches after 1-2 months of infection5. If the disease was left untreated, it could be lethal by renal failure or bladder cancer (S. haematobium) and acute hepatitis or liver fibrosis (S. mansoni)6.

Long-term schistosomiasis contributes to poverty. Poverty reduces water use options and increases risk of schistosomal infection7.

Many migrants and tourists come back from Africa after being infected with schistosomiasis through contact with contaminated fresh water leading to human schistosomiasis to be an emerging threat for Europe.8, 9

1.2 Life cycle of schistosome Schistosome has a very complex life cycle in both snail intermediate hosts and the human hosts. The life cycle begins with the elimination of eggs with feces or urine 2

from the infected human host. If the eggs come in contact with water, they release miracidia, which search for and penetrate the suitable snail intermediate hosts. The miracidia develop into cercariae and leave the snails into the water. In human hosts, the cycle begins when cercariae penetrate the human skin. They migrate with the bloodstream to the liver and the mesenteric veins. After maturing into adult schistosomula, female worms deposit eggs which are moved to the intestine, bladder or ureters. The cycle starts again when these eggs are eliminated with feces or urine.1, 3, 10-

1.3 Global distribution

Schistosoma japonicum (Asia13, 14), S mansoni (Middle East15, Africa16-19 and

South America20) and S haematobium (Africa16, 21 and Middle East15) are of regional and global importance. Schistosomiasis used to be endemic in Southern European (Portugal and Greece) until its eradication in the 1960s22. However, in August 2013, a German patient developed a urinary schistosomiasis, which became the first autochthonous case in Europe.23 In 2014, more than 258 million people required treatment for schistosomiasis. Compared to 2013, there was an increase of 14 million people treated for schistosomiasis in 2014, mainly in African Region.24

1.4 Prevention

It is difficult to eliminate intermediate hosts (snails) in schistosomiasis endemic areas, due to the high cost of chemical snail control (molluscicides) and high reproduction rate of snails25. The available methods to prevent and control schistosomiasis include clean water supplies26, better sanitation27, education25 and chemotherapy28. Preventive chemotherapy (mass treatment) at high-risk populations can result in significant reduction of schistosomal infection.29 3

1.5 Clinical features

1.5.1. Cercarial dermatitis

Cercarial dermatitis, known as ‘swimmer's itch', is a re-emerging disease in

Europe.30 This disease is caused by penetration of the skin by larvae of schistosome.

Parasites can migrate from the skin to the central nervous system or lungs. Extremely severe infections cause limb swelling, fever or diarrhea. The therapy of cercarial dermatitis includes topical application of abirritant powders or systemic administration of antihistaminics or corticosteroids.31, 32

1.5.2. Katayama fever

Acute schistosomiasis (Katayama fever) is ascribable to an immunologic response to the antigens present during the maturation process of the schistosomal worm. The patient may experience a cough, fever and urticarial rash with eosinophilia and pulmonary infiltrates. As the therapy for Katayama fever, a single oral dose of praziquantel is given after the acute stage.33, 34

1.5.3. Genitourinary schistosomiasis

Genitourinary schistosomiasis (bilharziasis) is caused by Schistosoma haematobium, which is the only species that infects the genitourinary system. The clinical symptoms of the early stages (within three months after infection) include dysuria, hematuria, ureteritis, pyelitis, and cystitis cystica. In the later stage of schistosomiasis, the deleterious function of the kidneys, ureters and bladder may be lethal due to renal failure or bladder cancer.35-37

1.5.4. Intestinal schistosomiasis

Immunologic adverse responses to schistosomal eggs which were trapped in the intestinal wall were associated with intestinal schistosomiasis. If initial eosinophilic inflammation is not relieved, eventually granulomas would form around the eggs. The clinical symptoms range from abdominal pain, anemia, and diarrhea, to microulcerations, 4

pseudopolyps, and obstruction of the colon. In the late stage, hematemesis caused by bleeding from esophageal varices may be fatal.38, 39

1.5.5. Neuroschistosomiasis

Neuroschistosomiasis can occur at any stage of schistosomal infection.40 This disease refers to the schistosomal involvement of both the brain and the spinal cord.

Schistosoma haematobium and S. mansoni usually cause myelopathy, while S. japonicum causes encephalic disease.41 Praziquantel and corticosteroids are the conventional treatment of neuroschistosomiasis. Surgery might be used for severe cases.42 The clinical symptoms include focal CNS impairment, seizures, increased intracranial pressure or transverse myelitis.40, 43

1.5.6. Cardiopulmonary schistosomiasis

Schistosomiasis was considered one of the main causes of pulmonary arterial hypertension (PAH). It is a severe disease with symptoms such as dyspnea, syncope, and peripheral edema.44 Most of the cardiopulmonary schistosomiasis are related to the portal hypertension, caused by the blockage of embolized schistosomal eggs.45

1.6 Clinically used drug (Praziquantel)

Chemotherapy is currently the only way to control schistosomiasis because no vaccine exists against schistosomiasis. The intermediate hosts (snails) are not easy to eliminate.46 Praziquantel (PZQ) is a synthetic tetracyclic tetrahydroisoquinoline derivative.47 The average cost of one treatment is $ 0.248 . However, the estimate cost of the treatment in KwaZulu-Natal, South Africa is $ 7.3 per child. Personnel, consumables, transport and capital items comprise a significant proportion of the cost.49

Figure 1.1 Antischistosomal agents

1.6.1. Activity and Toxicity

PZQ is the only drug available for treatment of all forms of schistosomiasis on a large scale50, 51. 60-90% cure rate (no detectable eggs) can be reached when the patient is treated with a single 40 mg/kg oral dose of PZQ. Although this is an excellent result,

100% cure rate is rare. This number may be overestimated, because of the insensitive method.46 Furthermore, praziquantel is active against adult schistosomes, but it has no activity against the juvenile schistosomula, the young developmental stages of the parasite. This is a key factor in the frequently observed treatment ‘failures’ in areas endemic for schistosomiasis.52

Praziquantel was demonstrated to have low toxicity in mice, rats, rabbits, and dogs in the acute toxicity, chronic toxicity, and carcinogenicity tests.53 Clinical trials confirmed the safety of PZQ on vital organs. Some mild and transient side effects, such as headache, gastrointestinal disturbance, insomnia and myalgia, disappear within 24 hours.54

1.6.2. Mechanism of action (MOA)

The precise MOA of PZQ has not been fully identified.55 The proposed targets of

2+ 56 PZQ are the Ca ion channels , specifically, the Cavβ ion channel subunit of schistosome57. PZQ promotes the muscular paralysis and calcium ions influx in S. mansoni.58 Another mechanism of PZQ is the disruption of schistosomal tegument which exposes the parasite antigens at the worm’s surface.55, 59

1.6.3. Drug resistance Resistance to PZQ in Egypt60 and Senegal61 due to the intensive application is contentious62-64. Under the laboratory condition, S. mansoni has the potential to develop 7

drug resistance to PZQ. After three doses of 300 mg/kg of PZQ were given during 28 to

37 days of infection, 93% of the seventh passage of S. mansoni survived65. In the long run, lacking development of an alternative drug may have serious consequences since

PZQ is the only drug that is available on the market for mass treatment of schistosomiasis66, 67.

1.7 Aryl hydantoins and antischistosomal activity The introduction of PZQ in the 1980s led to decisions to abandon the development of many promising antischistosomal agents that were discovered in the same period. One of these was AH01 (also called Ro 13- 3978), the lead compound from a series of aryl hydantoins that were investigated in some detail at Hoffmann La-

Roche68-71.

Some characteristics of AH01 justify it as a compelling antischistosomal prototype. First, AH01 is easily synthesized in a one-step procedure68, 71. Second, AH01 has a good pharmacokinetic profile with an elimination half-life in excess of 40 hours in experimental animals. Third, as reported by Link and Stohler71, AH01 has high oral efficacy against all three major schistosome species – S. mansoni, S. haematobium and

S. japonicum in a range of animal models. AH01 had a single oral dose ED50 of 26 mg/kg in S. mansoni-infected mice (adult worms). In this same schistosome mouse model, PZQ is considerably less effective against adult S. mansoni, with reported ED50 values ranging from 172 to 202 mg/kg71, and it has no significant activity against juvenile stages of the parasite. In contrast, AH01 has significant activity against the juvenile stage of the parasite; in S. mansoni-infected mice (juvenile worms), AH01 had a single oral dose ED50 of 140 mg/kg. Moreover, AH01 is schistosome specific as this aryl hydantoin had no activity in mice infected with either the intestinal fluke E. caproni or with the liver fluke F. hepatica. Finally, in a preliminary in vivo acute toxicity experiment, 8

Link and Stohler71 observed no apparent toxicity following administration of a single

1250 mg/kg dose of AH01 to mice.

AH01 has no effect on ex vivo S. mansoni. Despite the high in vivo antischistosomal efficacy of AH01, this aryl hydantoin at concentrations up to 100 μg/ml

(345 μM) over an incubation period of 48 hours had no activity against adult S. mansoni.

Thus, ex vivo S. mansoni seems to tolerate very high concentrations of AH01. On host immunology, PZQ kills fewer adult S. mansoni worms in T-cell deprived mice than in immunologically intact controls72.

AH01 also has antiandrogenic properties. This series of aryl hydantoins produced antiandrogenic side effects in the host68, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. Keiser recently demonstrated73 that nilutamide, but not the three structurally diverse androgen receptor

(AR) antagonists flutamide, bicalutamide, and cyproterone acetate, has weak, but measurable, antischistosomal activity in S. mansoni-infected mice. As phylogenetic evidence indicates that schistosome species do not appear to have AR’s74, these data indicate that for aryl hydantoins, the structural requirements for antischistosomal efficacy and AR binding interactions are divergent.

In this dissertation, I have further studied the antischistosomal and antiandrogenic properties of AH01. Administration of 100 mg/kg oral dose of AH01 to adult and juvenile Schistosoma mansoni infected mice produced 95 and 64% total worm burden reductions, confirming its high activity against adult worms, and demonstrating that AH01 is also effective against juvenile infections. AH01 had no measurable interaction with the androgen receptor in a ligand competition assay. This work provides the foundation for future exploration and discovery of aryl hydantoins with low cytotoxicities and promising in vivo antischistosomal activities. 9

A number of AH01 analogs were designed and synthesized to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-AR interactions. Although many of the compounds were inactive or weakly active, AR11, AR43, AR62, and AR64 had shown worm burden reduction (WBR) values >90%.

It was also discovered that urea carboxylic acid UC00, the product formed by hydrolysis of the hydantoin substructure of AH01, has substantial in vivo antischistosomal efficacy. A single 100 mg/kg oral dose of UC00 administered to S. mansoni-infected mice reduced adult WBR by 67%. However, like AH01, UC00 at concentrations up to 100 μg/mL (325 μM) had no measurable effect on ex vivo S. mansoni. We also found that UC00 at concentrations up to 30 μM was not cytotoxic to rat skeletal myoblast L6 cells. Thus, it was suggested that the relatively high antischistosomal efficacy, low cytotoxicity, and good physicochemical properties (Log D of 3.1 and PSA of 78 Å) of UC00 make this urea carboxylic acid an attractive starting point for optimization. In this respect, we have synthesized a small library of urea carboxylic acid analogs that are now undergoing biological testing.

The symmetrical N,N’-diaryl urea AR33 had been tested in S. mansoni-infected mice. AR33 was a side product formed in the synthesis of the aryl hydantoins. A single oral dose of AR33 (100 mg/kg) administered to S. mansoni-infected mice reduced adult

WBR by 40%, data which indicate that this N,N’-diaryl urea is more effective than the lead N,N’-diaryl urea MMV66585275. These data provide the basis to move forward with further optimization of these N,N’-diaryl ureas. In vitro and in vivo antischistosomal activities were evaluated for the diaryl urea library.

Overall, we have investigated the synthesis and antischistosomal activity of three classes of compounds. Specifically, this dissertation is outlined as follows: 10

 Chapter 2 discusses antischistosomal and antiandrogenic properties of aryl

hydantoin AH01.

 Chapter 3 focuses on the SAR of the antischistosomal aryl hydantoin AH01.

 Chapter 4 presents the procedure for synthesis of a library of urea carboxylic

acid analogs.

 Chapter 5 lays out the in vitro and in vivo antischistosomal activities of diaryl

ureas analogs.

 Chapter 6 discusses the overall conclusions of this dissertation and future

direction.

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31. Kolářová, L.; Horák, P.; Skírnisson, K.; Marečková, H.; Doenhoff, M. Cercarial dermatitis, a neglected allergic disease. Clin. Rev. Allergy Immunol. 2013, 45, 63-74.

32. Horák, P.; Mikeš, L.; Lichtenbergová, L.; Skála, V.; Soldánová, M.; Brant, S. V.

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165-190.

33. Puylaert, C. A.; van Thiel, P.-P. Katayama Fever. N. Engl. J. Med. 2016, 374,

469-469.

34. Logan, S.; Armstrong, M.; Moore, E.; Nebbia, G.; Jarvis, J.; Suvari, M.; Bligh, J.;

Chiodini, P. L.; Brown, M.; Doherty, T. Acute schistosomiasis in travelers: 14 years' experience at the Hospital for Tropical Diseases, London. Am. J. Trop. Med. Hyg. 2013,

88, 1032-1034.

35. Shebel, H. M.; Elsayes, K. M.; Abou El Atta, H. M.; Elguindy, Y. M.; El-Diasty, T.

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36. Khalaf, I.; Shokeir, A.; Shalaby, M. Urologic complications of genitourinary schistosomiasis. World J. Urol. 2012, 30, 31-38.

37. Kildemoes, A. O.; Kjetland, E. F.; Zulu, S. G.; Taylor, M.; Vennervald, B. J.

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38. Betson, M.; Sousa-Figueiredo, J. C.; Rowell, C.; Kabatereine, N. B.; Stothard, J.

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39. Montes, M.; White, A. C.; Kontoyiannis, D. P. Symptoms of intestinal schistosomiasis presenting during treatment of large B cell lymphoma. Am. J. Trop. Med.

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40. Pittella, J. E. Neuroschistosomiasis. Brain Pathol. 1997, 7, 649-662. 15

41. Ross, A. G.; McManus, D. P.; Farrar, J.; Hunstman, R. J.; Gray, D. J.; Li, Y.-S.

Neuroschistosomiasis. J. Neurol. 2012, 259, 22-32.

42. Ferrari, T. C. A.; Moreira, P. R. R. Neuroschistosomiasis: clinical symptoms and pathogenesis. Lancet Neurol. 2011, 10, 853-864.

43. Nascimento-Carvalho, C. M.; Moreno-Carvalho, O. A. Neuroschistosomiasis due to Schistosoma mansoni: a review of pathogenesis, clinical syndromes and diagnostic approaches. Rev. Inst. Med. Trop. Sao Paulo 2005, 47, 179-184.

44. dos Santos Ferreira, R. D. C.; Bandeira, A. P.; Domingues, A. L. C.

Schistosomiasis-Associated Pulmonary Arterial Hypertension. In Diagnosis and

Management of Pulmonary Hypertension, Springer: 2015; pp 143-163.

45. Lapa, M.; Dias, B.; Jardim, C.; Fernandes, C. J.; Dourado, P. M.; Figueiredo, M.;

Farias, A.; Tsutsui, J.; Terra-Filho, M.; Humbert, M. Cardiopulmonary manifestations of hepatosplenic schistosomiasis. Circulation 2009, 119, 1518-1523.

46. Cioli, D.; Pica-Mattoccia, L.; Basso, A.; Guidi, A. Schistosomiasis control: praziquantel forever? Mol. Biochem. Parasitol. 2014, 195, 23-29.

47. Dömling, A.; Khoury, K. Praziquantel and schistosomiasis. Chem. Med. Chem.

2010, 5, 1420-1434.

48. Hotez, P. J.; Engels, D.; Fenwick, A.; Savioli, L. Africa is desperate for praziquantel. J.-Lancet 2010, 376, 496-498.

49. Maphumulo, A. A.; Gagai, S.; Lothe, A.; Zulu, N.; Zwane, D.; Kildemoes, A. M.;

Vennervald, B. J.; Munsami, M.; Gundersen, S.; Taylor, M. Estimating the cost of the mass treatment campaign for schistosomiasis in Ugu District, KwaZulu-Natal, 2012. Trop.

Med. Int. Health 2013.

50. Cioli, D.; Pica-Mattoccia, L. Praziquantel. Parasitol. Res. 2003, 90, S3-S9. 16

51. Doenhoff, M. J.; Cioli, D.; Utzinger, J. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Current opinion in infectious diseases 2008, 21, 659-667.

52. Pica‐Mattoccia, L.; Cioli, D. Praziquantel: too good to be replaced? 2012; p 309-

321.

53. Frohberg, H. Results of toxicological studies on praziquantel. Arzneim. Forsch.

1983, 34, 1137-1144.

54. Chen, M. Use of praziquantel for clinical treatment and morbidity control of schistosomiasis japonica in China: a review of 30 years’ experience. Acta Trop. 2005, 96,

168-176.

55. Aragon, A. D.; Imani, R. A.; Blackburn, V. R.; Cupit, P. M.; Melman, S. D.;

Goronga, T.; Webb, T.; Loker, E. S.; Cunningham, C. Towards an understanding of the mechanism of action of praziquantel. Mol. Biochem. Parasitol. 2009, 164, 57-65.

56. Greenberg, R. M. Are Ca 2+ channels targets of praziquantel action? Int. J.

Parasitol. 2005, 35, 1-9.

57. Kohn, A. B.; Anderson, P. A.; Roberts-Misterly, J. M.; Greenberg, R. M.

Schistosome calcium channel β subunits unusual modulatory effects and potential role in the action of the antischistosomal drug praziquantel. J. Biol. Chem. 2001, 276, 36873-

36876.

58. Doenhoff, M. J.; Cioli, D.; Utzinger, J. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Curr. Opin. Infect. Dis. 2008, 21,

659-667.

59. Harnett, W.; Kusel, J. Increased exposure of parasite antigens at the surface of adult male Schistosoma mansoni exposed to praziquantel in vitro. Parasitology 1986, 93,

401-405. 17

60. Ismail, M.; Botros, S.; Metwally, A.; William, S.; Farghally, A.; Tao, L.-F.; Day, T.

A.; Bennett, J. L. Resistance to praziquantel: direct evidence from Schistosoma mansoni isolated from Egyptian villagers. Am. J. Trop. Med. Hyg. 1999, 60, 932-935.

61. Doenhoff, M. J.; Kusel, J. R.; Coles, G. C.; Cioli, D. Resistance of Schistosoma mansoni to praziquantel: is there a problem? Trans. R. Soc. Trop. Med. Hyg. 2002, 96,

465-469.

62. Ismail, M.; Metwally, A.; Farghaly, A.; Bruce, J.; Tao, L.-F.; Bennett, J. L.

Characterization of isolates of Schistosoma mansoni from Egyptian villagers that tolerate high doses of praziquantel. Am. J. Trop. Med. Hyg. 1996, 55, 214-218.

63. Fallon, P.; Tao, L.; Ismail, M.; Bennett, J. Schistosome resistance to praziquantel: fact or artifact? Parasitol. Today 1996, 12, 316-320.

64. Cioli, D. Praziquantel: is there real resistance and are there alternatives? Curr.

Opin. Infect. Dis. 2000, 13, 659-663.

65. Fallon, P. G.; Doenhoff, M. J. Drug-resistant schistosomiasis: resistance to praziquantel and oxamniquine induced in Schistosoma mansoni in mice is drug specific.

Am. J. Trop. Med. Hyg. 1994, 51, 83-88.

66. Doenhoff, M. J.; Pica-Mattoccia, L. Praziquantel for the treatment of schistosomiasis: its use for control in areas with endemic disease and prospects for drug resistance. Expert Rev. Anti-Infect. Ther. 2006, 4, 199-210.

67. Wang, W.; Wang, L.; Liang, Y. Susceptibility or resistance of praziquantel in human schistosomiasis: a review. Parasitol. Res. 2012, 111, 1871-1877.

68. Bernauer, K.; Helmut, L.; Harro, S. antiandrogenic and schistosomicidal imidazolidine derivatives. US 4234736 A, Nov 18, 1980, 1980.

69. Forget, E.; Félix, H.; Notteghem, M.; Léger, N. Appréciation de l'activité de dérivés schistosomicides en microscopie électronique. Bull. Soc. Pathol. Exot. Ses Fil.

1982. 18

70. Hansen, J. B.; Hafliger, O. Determination of the dissociation constant of a weak acid using a dissolution rate method. J. Pharm. Sci. 1983, 72, 429-431.

71. Link, H.; Stohler, H. R. 3-Arylhydantoine, eine substanzklasse mit schistosomizider wirkung. Eur. J. Med. Chem. 1984, 19, 261– 265.

72. Sabah, A.; Fletcher, C.; Webbe, G.; Doenhoff, M. Schistosoma mansoni: reduced efficacy of chemotherapy in infected T-cell-deprived mice. Exp. Parasitol. 1985, 60, 348-

354.

73. Keiser, J.; Vargas, M.; Vennerstrom, J. L. Activity of antiandrogens against juvenile and adult Schistosoma mansoni in mice. J. Antimicrob. Chemother. 2010, 65,

1991-1995.

74. de Mendonça, R. L.; Escrivá, H.; Bouton, D.; Laudet, V.; Pierce, R. J. Hormones and nuclear receptors in schistosome development. Parasitol. Today 2000, 16, 233-240.

75. Ingram-Sieber, K.; Cowan, N.; Panic, G.; Vargas, M.; Mansour, N. R.; Bickle, Q.

D.; Wells, T. N.; Spangenberg, T.; Keiser, J. Orally active antischistosomal early leads identified from the open access malaria box. PLoS Neglected Trop. Dis. 2014, 8, e2610.

Chapter 2

Antischistosomal versus Antiandrogenic Properties of Aryl Hydantoin AH01

Abstract

In the early 1980s, the antischistosomal aryl hydantoin AH01 (Ro 13-3978), a close structural analogue of the androgen receptor antagonist nilutamide, was discovered.

Administration of 100 mg/kg oral doses of AH01 to mice infected with adult and juvenile

Schistosoma mansoni produced 95 and 64% total worm burden reductions, confirming its high activity against adult worms, and demonstrating that AH01 is also effective against juvenile infections. AH01 had no measureable interaction with the androgen receptor in a ligand competition assay, but it did block dihydrotestosterone-induced cell proliferation in an androgen-dependent human prostate cancer cell line. For AH01, nilutamide, and three closely related aryl hydantoin derivatives, there was no correlation between antischistosomal activity and androgen receptor interaction.

2.1. Introduction

Schistosomiasis is a neglected but widespread tropical disease caused by infection with parasitic blood flukes. Schistosoma mansoni, S. haematobium, and S. japonicumare the most widely distributed species and cause the highest disease burden, particularly in sub-Saharan Africa.1-3 Praziquantel (PZQ) is the only drug available for treatment of this disease, and it is active against adult schistosomes, but has little activity against the juvenile schistosomula, the young developmental stages of the parasite.2-4 Should serious praziquantel drug resistance arise, there are no viable alternatives to this drug. Even so, drug discovery for schistosomiasis has languished. 20

The introduction of PZ in 1982 likely led to decisions to abandon the development of a number of promising antischistosomal agents that were discovered in the same time period. One of these was AH01 (Ro 13-3978), the lead compound from a series of aryl hydantoins that were investigated in some detail at Hoffmann La-Roche.5-8 A number of these aryl hydantoins had high oral efficacy in schistosome animal models,8 but they also produced antiandrogenic side effects in the host,5 a not unexpected outcome given the close structural similarity of these aryl hydantoins to the antiandrogenic drug nilutamide (Figure 2.1.). We recently demonstrated9 that nilutamide, but not the three structurally diverse androgen receptor (AR) antagonists flutamide, bicalutamide, and cyproterone acetate, has antischistosomal activity. As phylogenetic evidence indicates that schistosome species do not appear to have ARs,10 these data led us to hypothesize that for aryl hydantoins and related heterocycles, the structural requirements for antischistosomal efficacy and AR binding interactions are divergent.

Based on the limited elucidation of the structure-activity-relationship of this aryl hydantoin compound series,8 we began to test this hypothesis by the synthesis8, 11 of

AH01, its imino isostere AH02, and two derivatives (AH03 and AH04) incorporating nitrile functional groups (present in bicalutamide and other antiandrogens) known to import high AR binding affinity.12 I now present my initial characterization of the antischistosomal vs. antiandrogenic properties of these four aryl hydantoins.

F3C O Nilutamide, X = NO2, Y = O AH01, X = F, Y = O NH X N AH02, X = F, Y = NH AH03, X = CN, Y = O AH04, X = CN, Y = NH Y

Figure 2.1. Aryl hydantoin structures.

2.2. Chemistry

AH01-AH04 were obtained using a previously reported procedure.

The synthesis of 380 mg of AH01 (Scheme 2.1.) to initiate biological studies was successfully carried out in one step. It was obtained in 66 % yield by high-temperature reactions of 1-fluoro-4-iodo-2-(trifluoromethyl)benzene and hydantoin with cuprous oxide in DMF. Although effective for a first delivery, some important areas for further improvement were identified. An improved synthesis was thus pursued to (1) minimize the side product; and (2) simplify the work up procedure; (3) improve yield.

The optimized synthesis produced 15.021 g of AH01 in 90 % yield. The new procedure involved an efficient way to remove copper using sg to provide the crude mixture without the formation of hydrolysis side product AR37. A successive recrystallization from ethanol/H2O and ethyl acetate/hexane provided AH01 with the required purity. The formation of dimethylamine was reduced by replacing the solvent

DMF with DMA.

Scheme 2.1.

o Reagents and conditions: (a) Cu2O, DMF or DMA, 160 C, 24-42 h.

2.3. Results and Discussion

2.3.1. Ligand competition scintillation proximity assay (Figure 2.2.)

Interaction of the aryl hydantoins with the AR was characterized by a ligand competition scintillation proximity assay13 using a hexahistadine-tagged AR-ligand binding domain (LBD). As expected, for the homologous DHT competition experiment,

13 we measured a low IC50 value of 76 nM, similar to the previously reported value of 57 nM. Nilutamide and AH03 had IC50 values of 7.4 and 1.5 µM, respectively, whereas the other aryl hydantoins had IC50 values > 27 µM. The binding competition observed for nilutamide and AH03 is consistent with previous data demonstrating that AR ligands with aryl nitro or nitrile functional groups can form ion-dipole interactions with R752 in the AR ligand-binding domain.12

2.3.2. LNCaP cell proliferation.

To put the AR ligand competition data in context, we assessed the effects of the aryl hydantoins on androgen-dependent LNCaP C-33 human prostate cancer cells.14 As expected, DHT significantly induced LNCaP cell proliferation, and this was blocked by the antiandrogen bicalutamide (p<0.0001) (Figure 2.3.). Similarly, AH01 and AH02 blocked DHT-induced LNCaP cell proliferation (p<0.001), but these compounds had no effect on basal cell growth. Conversely, nilutamide, AH03, and AH04 alone stimulated

LNCaP cell growth (p<0.001), and for nilutamide, addition of DHT decreased (p<0.05) cell proliferation. These data indicate that nilutamide, AH03, and AH04 are AR agonists in this cell line. This is consistent with previous data15 revealing that nilutamide acts as an AR agonist in LNCaP cells, presumably as a consequence of the T868A mutation in the ligand-binding domain of the AR expressed in this cell line. It is also possible that 25

DHT may trigger a shutoff mechanism after maximal cell proliferation, consistent with previous results that LNCaP cells can respond to androgens in a biphasic concentration- dependent response.16, 17 For these hydantoins, the apparent discrepancies between the ligand competition scintillation proximity assay and LNCaP cell proliferation data may also be due to differential antagonistic effect on tetrameric complex dissociation, AR dimerization, nuclear translocation and AR interaction with androgen response elements in LNCaP cells.18, 19 Thus, androgen-sensitive LNCaP C-33 cells may not be the ideal screen to assess the androgenic/antiandrogenic properties of aryl hydantoins.

2.3.3. In vivo antischistosomal activities against S. mansoni

In vivo (Table 2.1.) antischistosomal activities against S. mansoni were assessed as previously described.9 Single 100 mg/kg oral doses of AH01 and AH02 administered to

S. mansoni-infected mice on day 49 post-infection (adult worms) afforded respective total worm burden reductions of 95 and 93%, confirming the previously reported8 high activities (ED50s of 38 and 37 mg/kg for AH01 and AH02) of these compounds. In this

4 same schistosome mouse model, PZQ has an ED50 value of 172 mg/kg. Similarly, administration of 100 and 200 mg/kg doses of AH01 and AH02 to S. mansoni-infected mice on day 21 post-infection (juvenile worms) afforded respective total worm burden reductions of 64/88 and 51/88%; this clearly demonstrates that aryl hydantoins, unlike

PZ, have significant (p < 0.05) activity against the juvenile stage of the parasite. In contrast to the high in vivo efficacies and lack in vivo toxicity that we observed for AH01 and AH02, administration of single 200 mg/kg oral doses of AH03 and AH04 to S. mansoni-infected or uninfected mice led to the death of 60-100% of all animals within 4-

5 days post-treatment. The observed toxicity of these two aryl hydantoins, the lack of apparent in vivo toxicity for nilutamide in this same experiment,9 and the lack of 26

interaction of AH04 with the AR, suggests that the in vivo toxicity of these aryl hydantoin derivatives derives in part from androgen-independent mechanisms.

Table 2.1. Effects of single oral doses of AH01 and AH02 administered to mice harboring 21-day-old juvenile and 49-day-old adult S. mansoni infections Total worm Dose Mean number of worms Infection Compound burden (mg/kg) (SD) reduction (%) Vehicle control 1* - 27.9 ± 12.5 - Vehicle control 2* - 17.4 ± 5.1 - Vehicle control 3* - 24.8 ± 5.2 - 100 6.3 ± 4.3‡ 64† AH01 200 3.0 ± 1.9¶ 88†

100 8.5 ± 6.8‡ 51 AH02 200 3.3 ± 3.3¥ 88† Juvenile Nilutamide§ 100 - 5 200 - 22 AH01 100 1.5 ± 1.9¶ 95† AH02 100 1.3 ± 1.3‡ 93† Adult Nilutamide§ 100 - 31 PZ§ 100 - 18

*3% ethanol and 7% Tween 80 solubilizing vehicle ¶versus vehicle control 1 ‡versus vehicle control 2 ¥versus vehicle control 3 †p < 0.05 from the Kruskal-Wallis test comparing the medians of the responses between the treatment and control groups §data from reference 9

Figure 2.2. AR SPA ligand displacement assay

Figure 2.3. Effects of aryl hydantoins on DHT-induced LNCaP cell proliferation. Bars represent the mean ± SE of the average of 2-4 sets of independent duplicate experiments. Open bars: 10 µM compound alone; Filled bars: 10 µM compound + 10 nM DHT.

2.4. Summary

In summary, we confirmed the high antischistosomal efficacy of AH01 and demonstrated that this aryl hydantoin is also effective against juvenile infections.

Further, AH01 had no measurable interaction with the AR in a ligand competition assay, but it did block DHT-induced cell proliferation in androgen-dependent human prostate cancer LNCaP cells. For AH01, nilutamide, and the other aryl hydantoins, there was no correlation between antischistosomal activity and androgen receptor interaction.

Although previous investigations6 demonstrate that AH01 produces schistosome worm lesions similar to those seen following PZ treatment, the molecular mechanism of action of this or related aryl hydantoins is unknown. However, based on our data, their antischistosomal properties do not seem to derive from interactions with the host AR.

Finally, it is interesting to note that although schistosome species do not appear to have

ARs10, Fantappié et al.20 discovered an expressed schistosome gene encoding the mitochondrial enzyme NADH ubiquinone oxidoreductase subunit 5 by screening a S. mansoni cDNA library with a human AR cDNA probe. This mitochondrial enzyme is thought to underlie the observed inhibition of S. mansoni growth by high concentrations of testosterone and dehydroepiandresterone.20, 21

2.5. Experimental

2.5.1. General.

Melting points are uncorrected. 1D 1H and 13C NMR spectra were recorded on a 500

MHz spectrometer using CDCl3 or DMSO-d6 as solvents. All chemical shifts are reported

1 in parts per million (ppm) and are relative to internal (CH3)4Si (0 ppm) for H and CDCl3

13 (77.2 ppm) or DMSO-d6 (39.5 ppm) for C NMR. EI GC-MS data were obtained using a 31

quadrapole mass spectrometer with 30 m DB-XLB type columns and a He flow rate of 1 mL/min. Silica gel (sg) particle size 32−63 μm was used for all flash column chromatography. Reported reaction temperatures are those of the oil bath.

2.5.2. Synthesis

3-[4-fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethylimidazolidine-2,4-dione (AH01)

Old method

To a solution of 5,5-dimethylhydantoin (338 mg, 2.64 mmol) and Cu2O (143 mg, 1.00 mmol) in DMF (10 mL) was added 1-fluoro-4-iodo-2-(trifluoromethyl)benzene (580 mg,

2.00 mmol). After it was heated under reflux for 24 h, the solvent was evaporated in vacuo. After addition of ammonium hydroxide (10 mL) and H2O (10 mL), the resulting precipitate was filtered and purified by chromatography (silica gel, hexane:EtOAc, 2:1) to afford 3-[4-fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethylimidazolidine-2,4-dione as a white solid (380 mg, 66%).

New method

To a solution of 5,5-dimethylimidazolidine-2,4-dione (9.70 g, 76 mmol) and Cu2O

(4.10 g, 29 mmol) in DMA (280 mL) was added 1-fluoro-4-iodo-2-

(trifluoromethyl)benzene (16.60 g, 57 mmol).The reaction mixture was heated at 160 oC for 42 h and cool to rt. After addition of ethyl acetate (250 mL), the resulting precipitation

(copper) was removed by filtration. The solvent was then filtered through silica gel to remove copper and the resulting cake was washed with ethyl acetate (100 mL).

Evaporation of the filtrate gave a crude product which was further purified by successive crystallization from a mixture of ethanol (20 mL) and H2O (500 mL) and a mixture of ethyl 32

acetate (10 mL) and hexane (200 mL) to afford 3-[4-fluoro-3-(trifluoromethyl)phenyl]-5,5- dimethylimidazolidine-2,4-dione as a white solid (15.021 g, 90 %).

1 H NMR (500 MHz, DMSO-d6) δ 1.41 (s, 6H), 7.66 (t, J = 9.7 Hz, 1H), 7.81 (ddd, J =

7.5, 4.5, 2.7 Hz, 1H), 7.90 (dd, J = 6.6, 2.5 Hz, 1H), 8.66 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 24.59, 57.95, 116.72 (qd, J = 32.9, 13.4 Hz),

117.70 (d, J = 21.7 Hz), 122.24 (q, J = 272.5 Hz), 125.68 (q, J = 4.8 Hz), 128.90 (d, J =

3.3 Hz), 133.55 (d, J = 9.2 Hz), 153.69, 157.56 (dd, J = 254.6, 2.4 Hz), 176.10.

GC-MS purity (Figure 2.4., Figure 2.5.): 100 %

Abundance

TIC: WCK160409_05.D\ data.ms 7000000 10.071

6000000

5000000

4000000

3000000

2000000

1000000

4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 Time-->

Figure 2.4. GC spectrum of AH01

Abundanc e

Scan 1133 (10.057 min): W CK160409_05.D\ data.ms 205.1 1100000

1000000

900000 290.1

800000

700000

600000

500000

400000

300000 57.2 84.1

200000 177.1 271.1 108.1 100000 138.0 157.1 226.0 246.1 0 60 80 100 120 140 160 180 200 220 240 260 280 m/ z-->

Figure 2.5. MS spectrum of AH01 35

2.5.3. Ligand competition scintillation proximity assay

To each well of a 96-well Ni-chelate-coated Flashplate® was added 80 μL of 5 μM

AR-LBD in the assay buffer (25 mM HEPES, pH 7.2, 50 mM NaCl, 0.01% NP-40, 20 µM dihydrotestosterone (DHT), 2% DMSO). After 60 min incubation, the protein solution was discarded followed by washes with assay buffer. Then, 40 μL of serial diluted aryl hydantoins in assay buffer containing 10% DMSO were added into each well followed by addition of 40 μL of 20 nM [3H]-DHT in assay buffer. The final assay solutions contained

5% DMSO. After sealing the plates and equilibrating for 18 h at 4 °C, radiocounts were measured and IC50 values were calculated.

2.5.4. LNCaP cell proliferation.

LNCaP cells (passage number between 39 to 42) were plated at a density of 5×104 or 6×104 cells/well in 6-well plates for 72 h in a regular RPMI 1640 culture medium containing 5% fetal bovine serum (FBS) which was maintained at 37 °C in a humidified atmosphere of 5% CO2. This was followed by continued cell culture for 48 h in a steroid- reduced medium consisting of phenol red-free RPMI 1640 containing 5% charcoal/dextran-treated FBS (v/v), 2 mM glutamine, and 50 μg/mL gentamicin. Cells were then exposed for an additional 72 h to 10 µM test compounds (from 10 mM DMSO stock solutions) with or without DHT (10 nM) after which time cell numbers were counted and cell growth relative to that of control cells was determined.

2.5.5. In vivo antischistosomal activities against S. mansoni

Mice were infected with ca. 80 S. mansoni cercariae on day 0 followed by administration of single 100 and 200 mg/kg oral doses of test compounds dissolved or suspended in a solubilizing 3% ethanol and 7% Tween 80 vehicle to groups of four to 36

five mice on day 21 post-infection (juvenile stage) and on day 49 post-infection (adult stage). At 28 days post-treatment, animals were sacrificed by the CO2 method and dissected to assess total worm reductions. 37

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14. Igawa, T.; Lin, F. F.; Lee, M. S.; Karan, D.; Batra, S. K.; Lin, M. F. Establishment and characterization of androgen ‐ independent human prostate cancer LNCaP cell model. Prostate 2002, 50, 222-235.

15. Veldscholte, J.; Berrevoets, C.; Ris-Stalpers, C.; Kuiper, G.; Jenster, G.;

Trapman, J.; Brinkmann, A.; Mulder, E. The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J Steroid Biochem Mol Biol 1992, 41, 665-669.

16. Olea, N.; Sakabe, K.; Soto, A. M.; Sonnenschein, C. The proliferative effect of

“anti-androgens” on the androgen-sensitive human prostate tumor cell line LNCaP.

Endocrinology 1990, 126, 1457-1463.

17. de Launoit, Y.; Veilleux, R.; Dufour, M.; Simard, J.; Labrie, F. Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells. Cancer Res 1991, 51, 5165-5170. 39

18. Veldscholte, J.; Berrevoets, C. A.; Brinkmann, A. O.; Grootegoed, J. A.; Mulder,

E. Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation.

Biochemistry 1992, 31, 2393-2399.

19. Georget, V.; Térouanne, B.; Nicolas, J.-C.; Sultan, C. Mechanism of antiandrogen action: key role of hsp90 in conformational change and transcriptional activity of the androgen receptor. Biochemistry 2002, 41, 11824-11831.

20. Fantappié, M. R.; Galina, A.; de Mendonça, R. L.; Furtado, D. R.; Secor, W. E.;

Colley, D. G.; Corrêa-Oliveira, R.; Freeman Jr, G.; Tempone, A. J.; De Camargo, L. L.

Molecular characterisation of a NADH ubiquinone oxidoreductase subunit 5 from

Schistosoma mansoni and inhibition of mitochondrial respiratory chain function by testosterone. Mol Cell Biochem 1999, 202, 149-158.

21. Morales-Montor, J.; Mohamed, F.; Ghaleb, A. M.; Baig, S.; Hallal-Calleros, C.;

Damian, R. T. In vitro effects of hypothalamic-pituitary-adrenal axis (HPA) hormones on

Schistosoma mansoni. J Parasitol 2001, 87, 1132-1139.

Chapter 3

Revisiting the SAR of the Antischistosomal Aryl Hydantoin AH01

Abstract

The aryl hydantoin Ro 13-3978 (AH01) was identified in the early 1980's as a promising antischistosomal lead compound. However, this series of aryl hydantoins produced antiandrogenic side effects in the host, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. Building on the known

SAR of this compound series, we now describe a number of analogs of AH01 designed to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-androgen receptor interactions. The principal SAR insight (Figure 3.1.) was that the hydantoin core of AH01 is required for high antischistosomal activity. We identified several compounds with high antischistosomal efficacy that were less antiandrogenic than AH01. These data provide direction for the ongoing optimization of antischistosomal hydantoins.

Figure 3.1. Principal SAR of AH01

3.1. Introduction

The introduction of Praziquantel (PZQ) in 1982 likely led to decisions to abandon the development of a number of promising antischistosomal agents that were discovered in the same time period. One of these was AH01 (Figure 3.2.), the lead compound from a series of aryl hydantoins that were investigated in some detail at F. Hoffmann La-

Roche1-4. As reported by Link and Stohler4, AH01 has high oral efficacy against all three major schistosome species – S. mansoni, S. haematobium and S. japonicum in a range

5 of animal models. Confirming these data, we found that AH01 had single oral dose ED50 values of 15 and 140 mg/kg against adult and juvenile S. mansoni in a mouse model. In this same schistosome mouse model, PZQ is considerably less effective against adult S.

4 mansoni, with reported ED50 values ranging from 172 to 202 mg/kg , and it has no significant activity against juvenile stages of the parasite. Despite the high in vivo antischistosomal efficacy of AH01, we found that this aryl hydantoin at concentrations up to 170 µM had almost no effect on cultured adult S. mansoni5. Data generated so far indicate that active metabolites do not account for the striking difference between the in vitro and in vivo antischistosomal activity of AH015.

However, this series of aryl hydantoins produced antiandrogenic side effects in the host1, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. We recently demonstrated6 that nilutamide, but not the three structurally diverse androgen receptor (AR) antagonists flutamide, bicalutamide, and cyproterone acetate, has weak, but measurable, antischistosomal activity in S. mansoni-infected mice. As phylogenetic evidence indicates that schistosome species do not appear to have AR’s7, these data led us to hypothesize that for aryl hydantoins and related heterocycles, the structural requirements for antischistosomal efficacy and AR binding interactions are divergent. 43

Figure 3.2. Praziquantel (PZQ), aryl hydantoins AH01 (Ro 13-3978) and nilutamide.

3.2. Design

Highlights from the SAR of this aryl hydantoin compound series conducted by

4 Roche are: 1) a combination of halogens (F, Cl) and/or CF3 groups at positions 3 and 4 of the phenyl ring were optimal; 2) EDG groups such as methoxy and dimethylamino at these same positions diminished activity; 3) 4-imino derivatives were active; and 4) some N1-substituted analogs were active.

Building on this foundation, we now describe a number of AH01 analogs (Tables

3.1.-3.4.) designed to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-AR interactions. For example, several target compounds maintain the 5,5-dimethylhydantoin core of AH01 and involve incorporation of sulfonamide (AR57 and AR17), phenethyl (AR18), aromatic halogens (AR06, AR27, AR31) and C=N bonds (AR04, AR08, AR43, AR48, AR52,

AR53, AR55), functional groups and structural elements demonstrated to abolish or diminish AR ligand affinity8-12. The final set of target compounds maintain the 4-fluoro-3- trifluoromethylphenyl substructure of AH01 in heterocycle variants of the 5,5- dimethylhydantoin substructure: succinimide AR16, urea AR36, oxolactam AR56, oxazolidinedione AR23, ring-expanded hydantoins AR32 and AR51, and hydantoin transpositional isomer AR58. Hydantoins analogous to AR58 had relatively weak AR binding affinity8.

Table 3.1. Antiandrogenic, and antischistosomal data for N1-substituted 4-fluoro-

3-trifluoromethylphenyl hydantoins

F3C O R N F N

f g Compd R LAPC4IC50 (µM) S. mansoni WBR (%) x 100 mg/kg po

AH01 H 4.4 95h

AR11 CH3 1.3 93

AR62 CH2CH3 0.14 98

AR63 CH2CONH2 agonist 25

AR13 CH2CN 0.29 91

AR18 (CH2)2C6H5 2.4 54

AR57 SO2CH3 6.3 19

AR17 SO2C6H5 4.3 44

fCells were then exposed to 10 nM DHT for 24 h in the presence of varying concentrations of test compounds. gGroups of five S. mansoni-infected NMRI mice were treated on day 49 post-infection with compounds dissolved or suspended in SSV. At 28 days post-treatment, animals were sacrificed and dissected to assess total worm burden reduction (WBR). p<0.05 from the Kruskal–Wallis test comparing the medians of the responses between the treatment and control groups. hdata from Keiser et al5.

Table 3.2. Antiandrogenic, and antischistosomal data for N3-substituted aryl hydantoins

f Compd X LAPC4IC50 (µM) S. mansoni WBR (%)1 x 100 mg/kg pog

h Nilutamide NO2 0.60/ 0.45 31

AR05 H 6.0 80

AR06 Cl 1.3 75i

AR59 CH3 >10 4.8 AR22 CN 3.7 10

AR54 CON(CH3)2 9.1 30

AR64 CF2H ≥10 94 AR53 H 3.8 1.8

AR43 CF3 4.5 100 AR55 1.3 17 AR08 H 9.6 38

AR52 CF3 >10 0 AR48 >10 42 AR28 4.2 56 a-gsee Table 3.1.footnotes. hdata from Keiser et al.6 i1/4 mice died

Table 3.3. Antiandrogenic, and antischistosomal data for 5-substituted 4-fluoro-3- trifluoromethylphenyl hydantoins

f g Compd X, Y LAPC4IC50 (µM) S. mansoniWBR (%)1 x 100 mg/kg po AR07b H, H 1.8 0

AR19b CH3, H >10 66

AR34 C6H5, H 5.3 41

AR40 CH3, 4.8 0

CH2OH

AR29 (CH2)4 0.99 61

AR30 (CH2)5 4.2 42 AR35 ------3.5 0 a-gsee Table 1 footnotes.

Table 3.4. Antiandrogenic, and antischistosomal data for 4-fluoro-3- trifluoromethylphenyl hydantoinheterocyle variants.

f g Compd LAPC4IC50 (µM) S. mansoni WBR (%)1 x 100 mg/kg po AR16 0.91 42 AR23 2.1 11 AR56 >10 0 AR36 8.1 59 AR58 2.8 0 AR32 >10 51h AR51 6.9 19 AR37 3.1 67

a-gsee Table 1 footnotes.

3.3. Chemistry

N1-alkyl and aralkyl hydantoins AR11, AR62, AR63, AR13, AR18 and sulfonamides AR57 and AR17 were obtained by N-alkylation and sulfonylation of AH01 according to the methods of Van Dort and Jung12 and Jung et al.8 (Scheme 3.1.).

Carboxyurea AR37 was obtained in high yield by hydrolysis of AH01 with aq. NaOH followed by acidification with dilute HCl.

Target compounds AR59, AR22, AR54, AR43, AR55, AR52, and AR48 were obtained in variable yields by high-temperature reactions of the corresponding aryl iodides (4-iodo-1-methyl-2-(trifluoromethyl)benzene, 2-fluoro-5-iodobenzonitrile, 2-fluoro-

5-iodo-N,N-dimethylbenzamide, 4-iodo-2-(trifluoromethyl)pyridine, 4-iodoquinoline, 5- iodo-2-(trifluoromethyl)pyridine and 2-iodopyrazine) and hydantoin (5,5- dimethylimidazolidine-2,4-dione) with cuprous oxide13, 14 in DMA (Scheme 3.2.).

Reactions between aryl iodide (1-fluoro-4-iodo-2-(trifluoromethyl)benzene) and spirohydantoin (1,3-diazaspiro[4.5]decane-2,4-dione) or succinimide (3,3- dimethylpyrrolidine-2,5-dione) under these same conditions afforded AR30 and AR16, respectively (Scheme 3). Similarly, AR64 was obtained by copper-catalyzed coupling of aryl bromide (4-bromo-2-(difluoromethyl)-1-fluorobenzene) and hydantoin (5,5- dimethylimidazolidine-2,4-dione), whereas AR28 was obtained by alkylation of benzyl bromide (4-(bromomethyl)-1-fluoro-2-(trifluoromethyl)benzene) with hydantoin (5,5- dimethylimidazolidine-2,4-dione) (Scheme 3.4.). Compound AR36 was obtained in a palladium-catalyzed N-arylation reaction15 between aryl iodide (1-fluoro-4-iodo-2-

(trifluoromethyl)benzene) and imidazolidin-2-one (4,4-dimethylimidazolidin-2-one)

(Scheme 3.3.)16.

N-arylation via copper (II) acetate promoted cross-coupling17-19 of boronic acids

(pyridin-3-ylboronic acid and pyridin-4-ylboronic acid) with hydantoin (5,5- 50

dimethylimidazolidine-2,4-dione) afforded AR08 and AR53; the same cross-coupling reaction between boronic acid ((4-fluoro-3-(trifluoromethyl)phenyl)boronic acid) and 1,3- oxazolidine-2,4-dione or bicyclic hydantoin (tetrahydro-1H-pyrrolo[1,2-c]imidazole-

1,3(2H)-dione) afforded AR23 and AR35, respectively (Scheme 3.5.).

Compounds AR19B, AR34, AR40 and AR29 were obtained in a two-step sequence4, 11, 20 by reactions between aryl isocyanate (1-fluoro-4-isocyanato-2-

(trifluoromethyl)benzene) and α-amino acids (alanine, (R)-2-amino-2-phenylacetic acid,

2-amino-3-hydroxy-2-methylpropanoic acid, and 1-aminocyclopentane-1-carboxylic acid) in aq. NaOH to form the corresponding urea carboxylic acids that then cyclized to the hydantoins when exposed to 2-4 M HCl at 110 °C (Scheme 3.6.). Compounds AR32 and AR51 were obtained by parallel two-step reactions between isocyanate (1-fluoro-4- isocyanato-2-(trifluoromethyl)benzene) and-amino acids (3-amino-3-methylbutanoic acid and 3-amino-2,2-dimethylpropanoic acid). In some instances, the acyclic urea carboxylic acid reaction intermediates precipitated from the initial reactions after acidification, but these were not purified, and were converted directly to the desired hydantoin reaction products. For almost all of these reactions, small amounts of the insoluble symmetrical N,N-diarylurea derived from isocyanate (1-fluoro-4-isocyanato-2-

(trifluoromethyl)benzene) were formed.

Oxolactam AR56 was obtained by cyclization of the anion of benzyl amide ester

(methyl 2-(2-(4-fluoro-3-(trifluoromethyl)phenyl)acetamido)-2-methylpropanoate) in a

Dieckmann-type condensation21; it was obtained from the corresponding acid chloride

(2-(4-fluoro-3-(trifluoromethyl)phenyl)acetyl chloride) (Scheme 3.7.).The key intermediate in the synthesis of AR58 was gem-dimethyl α-amino amide (2-((4-fluoro-3-

(trifluoromethyl)phenyl)amino)-2-methylpropanamide), which was obtained in a one-pot free radical multicomponent reaction22 from aniline (4-fluoro-3-(trifluoromethyl)aniline). 51

Exposure of this amino amide to 2,6-diisopropylphenyl isocyanate at high temperature23 effected carbonylativering closure to hydantoin AR58. Compounds AH01, AR05, AR06, and AR07B were synthesized following procedures described by Link et al4.

Scheme 3.1.a

aReagents and conditions: (a) NaH, DMF, rt, 0.5 h, then MeI, rt, 2h; (b) NaH, THF, 0 °C to rt, 3 h, then EtI, rt, 3d; (c) NaH, THF, 0 °C to rt, 3 h, then 2-bromoacetamide, rt, 3d; (d)

NaH, DMF, rt, 0.5 h, then 2-bromoacetonitrile, rt, 2h; (e) NaH, DMF, rt, 0.5 h, then 2- bromoethylbenzene, rt, 2h; (f) NaH, THF, 0 °C to rt, 0.5 h, then MeSO2Cl, rt, 3d; (g) NaH,

DMF, rt, 0.5 h, then benzenesulfonyl chloride, rt, 2 h; (h) 2 M NaOH, rt, 4 h, then 2 M

HCl.

Scheme 3.2.a

a Reagents and conditions: (a) Cu2O, DMA, 140-160 °C; 12-72 h.

Scheme 3.3.a

a Reagents and conditions: (a) Cu2O, DMA, 160 °C; 24 h; (b) Cu2O, DMF, 160 °C; 48 h

(29); (c) Pd2(dba)3, Xanthphos, Cs2CO3, toluene, 90 °C; 12 h (32).

Scheme 3.4.a

a Reagents and conditions: (a) Cu2O, DMA, 160 °C, 24 h; (b) K2CO3, DMA, 85 °C; 24 h.

Scheme 3.5.a

a Reagents and conditions: (a) Cu(OAc)2, MeOH, O2, 70 °C; 12 h; (b) Cu(OAc)2, pyridine,

CH2Cl2, rt, 7 d.

Scheme 3.6.a

(a) 1-2 M NaOH, 0 °C-rt, 3-12 h; (b) 2-4 M HCl, 110 °C, 2-12 h.

Scheme 3.7.a

(a) 1 M TiCl4 in CH2Cl2, formamide, acetone, 0 °C, 1 h, then Zn, 50% H2O2 in formamide,

0 °C, 3 h; (b) 2,6-diisopropylphenyl isocyanate, toluene, 250 °C, 5 bar, 10 min, MW; (c) methyl 2-amino-2-methylpropanoate HCl, Et3N, THF, rt, 12 h; (d) NaH, THF, rt, 12 h, then aq. AcOH.

3.4. Results and Discussion

3.4.1 Antiandrogenic and antischistosomal activities

We now consider the in vitro antiandrogenic and in vivo antischistosomal properties of these AH01 analogs. The former was assessed by inhibition of dihydrotestosterone (DHT)-dependent cell growth in the LAPC4 cell line, a cell line with a wild-type androgen receptor (AR), and the latter by measuring worm burden reduction

(WBR) in S. mansoni-infected mice. Contrary to our expectation based on the previous

SAR for this compound class, we did not observe decreased antiandrogenic potencies for sulfonamides AR57 and AR17 and phenethyl AR18 (Table 3.1.). N1-Substitution with small alkyl (AR11, AR62) groups increased antiandrogenic potencies, most strongly for the latter. Incorporation of nitrile (AR13) and carboxamide (AR63) functional groups into the N1-alkylsubstructure had very different effects; the former was a potent antiandrogen whereas the latter had no antiandrogenic properties and was instead a weak AR agonist.

Similar to that of AH01, N1-substituted hydantoins AR11, AR62, and AR13 had high antischistosomal activities; of the remaining compounds in this series, only AR18 had significant, but weak antischistosomal activity.

The antiandrogenic and antischistosomal properties of the N3-substituted aryl hydantoins exhibit several interesting trends (Table 3.2.). Replacing the 4-F in AH01 with a NO2 (Nilutamide) or Cl (AR06) increased antiandrogenic potency, whereas a H

(AR05) or Me (AR59) at this same position decreased antiandrogenic potency. Of these, hydantoins AR05 and AR06 had high antischistosomal activities, similar to previously reported data4. Replacing the 3-trifluoro in AH01 with a nitrile (AR22) had no effect on antiandrogenic potency and abolished antischistosomal activity. However, replacing the 3-trifluoromethyl in AH01 with a tertiary carboxamide (AR54) or 60

difluoromethyl (AR64) decreased antiandrogenic potency, and the latter had high antischistosomal activity. Hydantoin AR28, the benzyl derivative of AH01, had similar antiandrogenic potency and significantly reduced antischistosomal activity compared to the latter. As we had anticipated based on known SAR for antiandrogenic hydantoins, we observed decreased antiandrogenic potencies for some of the derivatives with aromatic C=N bonds; these included pyridines AR08 and AR52 and pyrazine AR48.

However, the only one of the C=N containing hydantoins to have high antischistosomal activity was AR43, the 4-pyridyl derivative with a trifluoromethyl group.

As the data in Table 3.3. illustrate, our initial foray into the SAR of the 5-position of AH01 did not bear much fruit. The principle insight gained was to note that replacing one, but not both, of the methyl groups decreases antiandrogenic activity. Of these derivatives, only AR29 had significant antischistosomal activity, but this 5,5- spirocyclopentyl derivative also had relatively potent antiandrogenic activity. Bicyclic hydantoin AR35 reveals that connecting the 5 and N1-position with a pyrrolidine substructure completely abolished antischistosomal activity and offered no improvement in antiandrogenic activity. Finally, the data in Table 3.4. shows that the hydantoin core of AH01 is required for high antischistosomal activity. Of these, only cyclic urea AR36 and urea carboxylic acid AR37 had significant antischistosomal activity. The latter is the hydrolysis product of AH01 and is formed in small quantities when AH01 is administered at high doses. Interestingly, as reported by Link and Stohler4, the methyl ester of AR37 has good antischistosomal activity.

3.5. Summary

The SAR of the 5-substituted aryl hydantoins reveals that replacing one of the methyl groups with a hydrogen atom decreases antiandrogenic activity and retains 61

significant antischistosomal activity. Investigation of heterocycle variants showed that the hydantoin core of AH01 is required for high antischistosomal activity.

3.6. Experimental Section

3.6.1. General.

Melting points are uncorrected. 1D 1H and 13C NMR spectra were recorded on a

500 MHz spectrometer using CDCl3 or DMSO-d6 as solvents. All chemical shifts are

1 reported in parts per million (ppm) and are relative to internal (CH3)4Si (0 ppm) for H

13 and CDCl3 (77.2 ppm) or DMSO-d6 (39.5 ppm) for C NMR. EI GC-MS data were obtained using a quadrapole mass spectrometer with 30 m DB-XLB type columns and a

He flow rate of 1 mL/min.Silica gel (sg) particle size 32−63 μm was used for all flash column chromatography. Reported reaction temperatures are those of the oil bath.

5,5-Dimethyl-3-(pyridin-3-yl)imidazolidine-2,4-dione (AR08).

To a solution of 5,5-dimethylimidazolidine-2,4-dione (1.792 g, 14mmol) and Cu(OAc)2

(181 mg, 1mmol) in methanol (50 mL) was added pyridin-3-ylboronic acid (1.23 g, 10

o mmol) under O2.The mixture was heated at 70 C overnight. The solvent was then filtered through silica gel to remove copper and the resulting cake was washed with methanol (100 mL). Evaporation of the filtrate gave a crude product which was further purified by successive crystallizations from diethyl ether (10 mL) and saturated Na2CO3

(10 mL) to afford AR08 as a white solid (1.258 g, 61%). mp 160–162 °C; 62

1 H NMR (CDCl3) δ 1.56 (s, 6H), 6.85 (s, 1H), 7.43 (dd, J = 4.4, 7.8 Hz, 1H), 7.84 (d, J =

8.3 Hz, 1H), 8.62 (d, J = 4.4 Hz, 1H), 8.78 (s, 1H);

13 C NMR (CDCl3) δ 25.22, 58.87, 123.57, 128.78, 133.15, 146.79, 148.65, 154.72,

175.79.

Anal Calcd for C10H11N3O2: C, 58.53; H, 5.40; N, 20.48; Found: C, 58.70; H, 5.36; N,

20.19.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-1,5,5-trimethylimidazolidine-2,4-dione (AR11)

To a solution of 3-(4-fluoro-3-(trifluoromethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione

(700 mg, 2.4 mmol) in DMF was added NaH (87 mg, 3.6 mmol) under Ar. The mixture was stirred at rt for 30 min and then iodomethane (514 mg, 3.6 mmol) was added dropwise. After stirring for 2 h, the mixture was evaporated in vacuo to give a residue which was extracted with brine (30 mL) and EtOAc (3 x 30 mL). The combined organic phase was washed with brine (2 x 30 mL) and dried over MgSO4. After removal of the solvents, the residue was purified by chromatography (sg, hexane:EtOAc, 4:1) to afford

AR11 as a white solid (580 mg, 79%). mp 110–112 °C;

1 H NMR (CDCl3) δ 1.51 (s, 6H), 2.98 (s, 3H), 7.29 (t, J = 9.3 Hz, 1H), 7.66-7.69 (m, 1H),

7.77(d, J = 6.3 Hz, 1H); 63

13 C NMR (CDCl3) δ 22.28, 24.68, 61.22, 117.48 (br), 118.96 (qd, J = 33.6, 13.9 Hz),

122.03 (q, J = 272.5 Hz), 124.80 (br), 128.07(d, J = 3.6 Hz), 131.23 (d, J = 8.6

Hz),153.44, 158.48 (q, J = 258.2 Hz), 175.14.

Anal Calcd for C13H12F4N2O2: C, 51.32; H, 3.98; N, 9.21; Found: C, 51.20; H, 3.92; N,

9.39.

2-[3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethyl-2,4-dioxoimidazolidin-1- yl]acetonitrile (AR13)

To a solution of 3-(4-fluoro-3-(trifluoromethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione

(700 mg, 2.4 mmol) in DMF was added NaH (116 mg, 4.8 mmol) under Ar. The mixture was stirred at rt for 30 min and then 2-bromoacetonitrile (578 mg, 4.8 mmol) was added dropwise. After stirring for 2 h, the mixture was evaporated in vacuo to give a residue which was extracted with brine (30 mL) and EtOAc (3 × 30 mL). The combined organic phase was washed with brine (2 × 30 mL) and dried over MgSO4. After removal of the solvents, the residue was purified by chromatography (sg, hexane:EtOAc, 2:1) to afford

AR13 as a white solid (573 mg, 72%). mp147–149 °C;

1 H NMR (CDCl3) δ 1.65 (s, 6H), 4.37 (s, 2H), 7.32 (t, J = 9.3 Hz, 1H), 7.66-7.69 (m, 1H),

7.75 (dd, J = 2.4, 5.9 Hz, 1H); 64

13 C NMR (CDCl3) δ 22.75, 27.14, 62.11, 114.73, 117.81 (d, J = 22.1 Hz), 119.28 (qd, J =

33.6, 13.9 Hz), 121.90 (q, J = 273.0 Hz), 124.89 (q, J = 1.9 Hz), 127.28 (d, J = 4.4 Hz),

131.27 (d, J = 9.6 Hz), 153.13, 158.83 (q, J = 257.2 Hz), 173.71.

Anal Calcd for C14H11F4N3O2: C, 51.07; H, 3.37; N, 12.76; Found: C, 51.29; H, 3.60; N,

12.52.

1-[4-Fluoro-3-(trifluoromethyl)phenyl]-3,3-dimethylpyrrolidine-2,5-dione (AR16 29)

To a solution of 3,3-dimethylpyrrolidine-2,5-dione (1.71 g, 13.5 mmol) and Cu2O (0.74 g,

5.2 mmol) in DMF (15 mL) was added 1-fluoro-4-iodo-2-(trifluoromethyl)benzene (3.0 g,

10.3 mmol) and the mixture was heated to reflux for 48 h. After cooling to rt, the solvent was evaporated in vacuo. After addition of conc. ammonium hydroxide (20 mL) and H2O

(15 mL), the resulting precipitate was filtered and purified by chromatography (sg, hexane:EtOAc, 5:1) to afford AR16 as a white solid (1.39 g, 46%). mp 134–136 °C;

1 H NMR (CDCl3) δ 1.44 (s, 6H), 2.74 (s, 2H), 7.33 (t, J = 9.3 Hz, 1H), 7.53-7.56 (m, 1H),

7.62(d, J = 5.9 Hz, 1H);

13 C NMR (CDCl3) δ 25.60, 40.23, 43.62, 117.67 (d, J = 22.1 Hz), 119.15 (qd, J = 34.6,

13.4 Hz), 121.95 (q, J = 272.5 Hz), 125.33 (br), 128.0 (d, J = 3.8 Hz), 131.9 (d, J = 8.6

Hz),158.89 (d, J = 258.6 Hz), 174.20, 181.67. 65

Anal Calcd for C13H11F4NO2: C, 53.99; H, 3.83; N, 4.84; Found: C, 54.00; H, 3.82; N,

4.89.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethyl-1-

(phenylsulfonyl)imidazolidine-2,4-dione (AR17).

To a solution of 3-(4-fluoro-3-(trifluoromethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione

(700 mg, 2.4 mmol) in DMF (15 mL) was added NaH (87 mg, 3.6 mmol) under Ar. The mixture was stirred at rt for 30 min and then benzenesulfonyl chloride (639 mg, 3.6mmol) was added dropwise. After stirring for 2 h, the mixture was evaporated in vacuo to give a residue which was extracted with brine (30 mL) and EtOAc (3 × 30 mL). The combined organic phase was washed with brine (2 × 30 mL) and dried over MgSO4. After removal of the solvents, the residue was purified by chromatography (sg, hexane:EtOAc, 4:1) to afford AR17 as a white solid (739 mg, 71%). mp 153–155 °C;

1 H NMR (CDCl3) δ 1.92 (s, 6H), 7.27 (t, J = 9.4 Hz, 1H), 7.57-7.71 (m, 5H), 8.13 (d, J =

8.3 Hz, 2H);

13 C NMR (CDCl3) δ 24.26, 66.75, 117.80 (d, J = 22.2 Hz),119.29 (qd, J = 34.1, 13.9Hz),

121.77 (q, J = 272.5 Hz), 125.14 (br), 126.46 (d, J = 3.8 Hz),128.73, 129.16, 131.54 (d, J

= 9.1 Hz), 134.59, 138.32, 150.57, 158.99 (d, J = 259.6 Hz), 172.81. 66

Anal Calcd for C18H14F4N2O4S·0.7H2O: C, 48.80; H, 3.50; N, 6.32; Found: C, 48.44; H,

3.09; N, 6.12.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethyl-1-phenethylimidazolidine-2,4- dione (AR18).

To a solution of 3-(4-fluoro-3-(trifluoromethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione

(700 mg, 2.4 mmol) in DMF (15 mL) was added NaH (87 mg, 3.6 mmol) under Ar. The mixture was stirred at rt for 30 min and then 2-bromoethylbenzene (670 mg, 3.6 mmol) was added dropwise. After stirring for 2 h, the mixture was evaporated in vacuo to give a residue which was extracted with brine (30 mL) and EtOAc (3 × 30 mL). The combined organic phase was washed with brine (2 × 30 mL) and dried over MgSO4. After removal of the solvents, the residue was purified by chromatography (sg, hexane:EtOAc, 4:1) to afford AR18 as a white solid (410 mg, 43%). mp 98–100 °C;

1 H NMR (CDCl3) δ 1.39 (s, 6H), 3.05 (t, J = 7.8 Hz, 2H), 3.53 (t, J = 8.3 Hz, 2H), 7.24-

7.34 (m, 6H), 7.69-7.72 (m, 1H), 7.78 (dd, J = 2.4, 6.9 Hz, 1H);

13 C NMR (CDCl3) δ 23.12, 35.34, 42.23, 61.86, 117.46 (d, J = 22.2 Hz),118.94 (qd, J =

33.1, 13.9Hz), 122.04 (q, J = 272.0 Hz), 124.72 (br), 126.81,128.69, 128.81, 131.14 (d, J

= 8.5 Hz),138.23, 153.62, 158.49 (d, J = 258.2 Hz), 174.94. 67

Anal Calcd for C20H18F4N2O2: C, 60.91; H, 4.60; N, 7.10; Found: C, 60.70; H, 4.89; N,

7.26.

5-(4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)-2-fluorobenzonitrile (AR22 12)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (0.674 g, 5.3 mmol) and Cu2O (290 mg, 2 mmol) in DMA (4 mL) was added 2-fluoro-5-iodobenzonitrile (1.0 g, 4.1 mmol).

After it was heated to 160 oC for 48 h, the solvent was evaporated in vacuo. After addition of EtOAc (100 mL), the resulting precipitate was filtered and the filtrate was concentrated and purified by chromatography (sg, EtOAc) followed by recrystallization from ethanol:H2O (1:20) to afford AR22 as a white solid (768 mg, 77%). mp 145–147 °C;

1 H NMR (CDCl3) δ 1.56 (s, 6H), 6.70 (s, 1H), 7.33 (t, J = 8.8 Hz, 1H), 7.78-7.81 (m, 1H),

7.84 (dd, J = 2.4, 5.4 Hz, 1H);

13 C NMR (CDCl3) δ25.15, 58.83, 102.08 (d, J = 17.3 Hz), 112.99, 117.08 (d, J = 21.1 Hz),

128.52 (d, J = 3.4 Hz), 130.48, 132.28 (d, J = 8.6 Hz), 154.41, 161.74 (d, J = 261.0 Hz),

175.47.

Anal Calcd for C12H10FN3O2: C, 58.30; H, 4.08; N, 17.00; Found: C, 58.08; H, 4.26; N,

17.28

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethyloxazolidine-2,4-dione (AR23). 68

To a solution of 5,5-dimethyloxazolidine-2,4-dione (903 mg, 7.0 mmol) and Cu(OAc)2 (90 mg, 0.5 mmol) in methanol (25 mL) was added (4-fluoro-3-

(trifluoromethyl)phenyl)boronic acid (1.04 g, 5.0mmol) under O2.The mixture was heated at 70 oC overnight. After cooling to rt, the solvent was evaporated in vacuo. The residue was purified by chromatography with successive elution with hexane and EtOAc followed by crystallization from ethanol:H2O (1:10) to afford AR23 as a white solid (421 mg, 29%). mp 174–176 °C;

1 H NMR (CDCl3) δ 1.70 (s, 6H), 7.34 (t, J = 8.8 Hz, 1H), 7.69-7.72 (m, 1H), 7.79 (dd, J =

2.4, 5.9 Hz, 1H);

13 C NMR (125.7 MHz, CDCl3) δ 23.74, 83.80, 117.99 (d, J = 22.1 Hz), 119.48 (qd, J =

34.1, 13.9 Hz), 121.81 (q, J = 272.5 Hz), 124.53 (br),127.12 (d, J = 3.4 Hz),130.89 (d, J

= 9.1 Hz),152.54, 159.01 (dq, J = 259.6, 1.9 Hz), 174.32.

Anal Calcd for C12H9F4NO3: C, 49.49; H, 3.12; N, 4.81; Found: C, 49.21; H, 3.06; N, 4.80.

3-[4-Fluoro-3-(trifluoromethyl)benzyl]-5,5-dimethylimidazolidine-2,4-dione (AR28)

A mixture of 4-(bromomethyl)-1-fluoro-2-(trifluoromethyl)benzene (1.552 g, 6.0 mmol),

5,5-dimethylimidazolidine-2,4-dione (647 mg, 5.1 mmol) and K2CO3 (2.004 g, 14.5 mmol) in DMA (12.5 mL) was stirred at 85 oC for 24 h. The mixture was extracted with

EtOAc (3 × 50 mL) and H2O (50 mL). The organic phase was combined, dried, and evaporated in vacuo to give a residue which was further purified by chromatography (sg,

EtOAc:hexane, 1:3) followed by crystallization from H2O (5 mL) to afford AR28 as a white solid (1.16 g, 75%). mp 86–87 oC;

1 H NMR (DMSO-d6) δ 1.32 (s, 6H), 4.63 (s, 2H), 7.50 (dd, J = 10.7, 8.6 Hz, 1H), 7.56-

7.63 (m, 1H), 7.66 (d, J = 7.0 Hz, 1H), 8.46 (s, 1H);

13 C NMR (DMSO-d6) δ 24.53, 39.91, 58.03, 116.53 (qd, J = 32.4, 12.6 Hz), 117.55 (d, J

= 20.7 Hz), 122.47 (q, J = 272.0 Hz), 126.07 (q, J = 4.7 Hz), 133.99, 134.04 (d, J = 5.3

Hz), 154.91, 158.10 (dq, J = 253.2, 2.3 Hz), 177.22.

Anal Calcd for C13H12F4N2O2: C, 51.32; H, 3.98; N, 9.21. Found: C, 51.49; H, 4.12; N,

9.34.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-1,3-diazaspiro[4.4]nonane-2,4-dione (AR29)

Step 1.To a solution of 1-aminocyclopentane-1-carboxylic acid (1.161 g, 9.0 mmol) in 1

M NaOH (15 mL) was added a solution of 1-fluoro-4-isocyanato-2-

o (trifluoromethyl)benzene (1.83 g, 8.9 mmol) in CH3CN (10 mL) at 0 C dropwise. The mixture was stirred at 0 oC for 3 h and then warmed to rt overnight. The reaction mixture 70

was adjusted to pH=1.0 by 32% HCl and concentrated in vacuo. Step 2. The resulting precipitate was collected by filtration and the solid was then suspended in 4 M HCl (50 mL) and heated at 110 °C overnight. After the reaction mixture was cooled to rt, the resulting precipitate was filtered and crystallized from diethyl ether to give AR29 as a white solid (660 mg, 23%). mp 151–152 oC;

1 H NMR (DMSO-d6) δ 1.67-1.92 (m, 6H), 2.01-2.16 (m, 2H), 7.65 (dd, J = 10.5, 9.0 Hz,

1H), 7.78-7.84 (m, 1H), 7.90 (dd, J = 6.6, 2.6 Hz, 1H), 8.86 (s, 1H);

13 C NMR (DMSO-d6) δ 24.82, 37.58, 67.49, 116.86 (qd, J =33.2, 13.4 Hz), 117.89 (d, J =

21.9 Hz), 122.43 (q, J = 272.4 Hz), 125.72, 129.14, 133.61 (d, J = 9.0 Hz), 154.16,

157.65 (d, J = 253.1 Hz), 176.55.

Anal Calcd for C14H12F4N2O2: C, 53.17; H, 3.82; N, 8.86. Found: C, 52.89; H, 4.00; N,

8.89.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-1,3-diazaspiro[4.5]decane-2,4-dione (AR30)

To a solution of 1,3-diazaspiro[4.5]decane-2,4-dione (840 mg, 5.0 mmol) and Cu2O (290 mg, 2.0 mmol) in DMA (20 mL) was added 1-fluoro-4-iodo-2-(trifluoromethyl)benzene

(1170 mg, 4.0 mmol). After it was heated at 160 oC for 24 h, the solvent was evaporated in vacuo. The residue was purified by chromatography (sg. EtOAc) to afford AR30 as a white solid (917 mg, 69%). mp 213–215 oC; 71

1 H NMR (DMSO-d6) δ 1.25-1.40 (m, 1 H) 1.58 (br s, 3H) 1.64-1.83 (m, 6H) 7.65 (t, J =

9.7 Hz, 1H), 7.77 – 7.84 (m, 1H) 7.89 (d, J = 6.35 Hz, 1H) 9.05 (br s, 1H);

13 C NMR (DMSO-d6) δ 20.93, 24.56, 33.33, 61.27, 116.88 (qd, J = 33.0, 13.3), 117.9 (d,

J = 22.0), 122.4 (q, J = 272.5), 125.87 (d, J = 4.1), 129.03, 133.76 (d, J = 9.2), 154.27,

157.7 (d, J = 254.7), 175.64.

Anal Calcd for C15H14F4N2O2: C, 54.55; H, 4.27; N, 8.48. Found: C, 54.38; H, 4.40; N,

8.49.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-6,6-dimethyldihydropyrimidine-2,4(1H,3H)- dione (AR32)

Step 1. To a solution of 3-amino-3-methylbutanoic acid (585 mg, 5.0mmol) in 2 M NaOH

(5 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (1.95 g, 9.5 mmol).

After it was stirred at rt for 4 h, the reaction mixture was filtered to remove the insoluble

N,N-diarylurea side product, and the filtrate was adjusted to pH=1.0 with 2 M HCl and concentrated. Step 2. The residue was suspended in 2 M HCl (50 mL) and then heated at 110 °C for 2 h. After the reaction mixture was cooled to rt, the resulting precipitate was filtered and washed with H2O (20 mL) to give AR32 as a white solid (477 mg, 31%). mp 225–226 oC;

1 H NMR (DMSO-d6) δ 1.31 (s, 6H), 2.74 (s, 2H), 7.53–7.64 (m, 2H), 7.71 (dd, J = 6.7,

2.4 Hz, 1H), 8.18 (s, 1H); 72

13 C NMR (DMSO-d6) δ 28.11, 44.32, 48.22, 116.88 (qd, J = 32.6, 13.3 Hz), 117.68 (d, J

= 21.5 Hz), 122.54 (q, J = 272.3 Hz), 128.60 (d, J = 4.8 Hz), 132.82 (d, J = 3.5 Hz),

136.61 (d, J = 9.1 Hz), 152.51, 158.16 (d, J = 254.0 Hz), 169.81.

Anal Calcd for C13H12F4N2O2·0.5 H2O: C, 49.85; H, 4.18; N, 8.94. Found: C, 49.79; H,

4.51; N, 8.81. GC-MS purity: 100%

(R)-3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5-phenylimidazolidine-2,4-dione (AR34)

Step 1. To a solution of (R)-2-amino-2-phenylacetic acid (1.057 g, 7.0 mmol) in 1 M

NaOH (10 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (2.05 g, 10

o o mmol) in CH3CN (5 mL) at 0 C dropwise. The mixture was stirred at 0 C for 3 h and then 1,4-dioxane (10 mL) was added according to the method of Cooper et al24. After stirring at rt overnight, the reaction mixture was adjusted to pH=2 by 32% HCl and concentrated in vacuo. Step 2. The resulting precipitate was successively crystallized from EtOH:H2O (1:10) and EtOAc:hexane (1:10) to give (R)-2-[3-[4-fluoro-3-

(trifluoromethyl)phenyl]ureido]-2-phenylacetic acid as a white solid (1.661 g, 67%), 818 mg of which was then suspended in 4 M HCl (50 mL) and heated at 110 °C overnight.

After the reaction mixture was cooled to 0 oC, the resulting precipitate was filtered to give

AR34 as a white solid (678 mg, 87%). mp 166–168 oC;

1 H NMR (DMSO-d6) δ 5.37 (s, 1H), 7.33-7.54 (m, 5H), 7.67 (dd, J = 10.0, 9.7 Hz, 1H),

7.78-7.88 (m, 1H), 7.92 (dd, J = 6.7, 2.5Hz, 1H), 9.10 (s, 1H). 73

13 C NMR (DMSO-d6) δ 60.36, 117.01 (qd, J =32.8, 13.5 Hz), 118.05 (d, J = 21.8 Hz),

122.40 (q, J = 272.7 Hz), 125.89, 127.55, 128.76, 128.89, 128.97 (d, J = 3.4 Hz), 133.80

(d, J = 9.3 Hz), 135.55, 155.33, 157.85 (d, J = 258.6 Hz), 171.60.

Anal Calcd for C16H10F4N2O2: C, 56.81; H, 2.98; N, 8.28. Found: C, 56.66; H, 3.12; N,

8.18.

2-[4-Fluoro-3-(trifluoromethyl)phenyl]tetrahydro-1H-pyrrolo[1,2-c]imidazole-

1,3(2H)-dione (AR35)

To a solution of tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione (538 mg, 3.8 mmol), Cu(OAc)2 (1.370 g, 7.6 mmol) and pyridine (900 mg, 11.4 mmol) in CH2Cl2 (38 mL) was added [4-fluoro-3-(trifluoromethyl)phenyl]boronic acid (2.359 g, 11.3 mmol).

After stirring at rt for 7 days, the solvent was removed in vacuo. The residue was purified by chromatography (sg, EtOAc) and then crystallized from hexane to afford AR35 as a white solid (443 mg, 39%). mp 146–148 oC;

1 H NMR (CDCl3) δ 1.78-1.93 (m, 1H), 2.06-2.30 (m, 2H), 2.32-2.45 (m, 1H), 3.37 (ddd, J

= 11.6, 8.5, 4.6 Hz, 1H), 3.80 (dt, J = 11.5, 7.8 Hz, 1H), 4.27 (dd, J = 9.4, 7.5 Hz, 1H),

7.29 (t, J = 9.3 Hz, 1H), 7.60-7.68 (m, 1H), 7.74 (dd, J = 6.2, 2.6 Hz, 1H);

13 C NMR (DMSO-d6) δ 26.38, 26.50, 45.30, 62.94, 116.77 (qd, J = 33.1, 13.5 Hz),

117.87 (d, J = 21.8 Hz), 122.20 (q, J = 272.0 Hz), 125.68, 128.89 (d, J = 3.4 Hz), 133.59

(d, J = 9.4 Hz), 157.66 (d, J = 255.0 Hz), 158.26, 172.45. 74

Anal Calcd for C13H10F4N2O2: C, 51.66; H, 3.34; N, 9.27. Found: C, 51.70; H, 3.40; N,

9.55.

1-[4-Fluoro-3-(trifluoromethyl)phenyl]-4,4-dimethylimidazolidin-2-one (AR36)

To a solution of 4,4-dimethyl-2-imidazolidinone (500 mg, 4.4 mmol), Cs2CO3 (2.460 g,

7.5 mmol), Xantphos (108 mg, 0.19 mmol) and Pd2(dba)3 (84 mg, 0.09 mmol) in toluene

(40 mL) was added 1-fluoro-4-iodo-2-(trifluoromethyl)benzene (1.270 g, 4.4 mmol). The reaction mixture was stirred at 90 oC for 12 h under Ar. Evaporation gave a crude product which was purified by washing with water (25 mL), chromatography (sg, EtOAc), and recrystallizion from hexane to afford AR36 as a white solid (939 mg, 77%). mp 147–148 oC;

1 H NMR (DMSO-d6) δ 1.28 (s, 6H), 3.64 (s, 2H), 7.39 (s, 1H), 7.45 (t, J = 9.8 Hz, 1H),

7.72 (dt, J = 9.2, 3.6 Hz, 1H), 8.05 (dd, J = 6.4, 2.9 Hz, 1H);

13 C NMR (DMSO-d6) δ 28.20, 50.57, 57.07, 114.69 (q, J = 5.2 Hz), 116.22 (qd, J = 32.1,

12.9 Hz), 117.29 (d, J = 21.3 Hz), 122.30 (d, J = 7.7 Hz), 122.66 (q, J = 272.3 Hz),

137.58, 153.26 (d, J = 247.5 Hz), 157.02.

Anal Calcd for C12H12F4N2O: C, 52.18; H, 4.38; N, 10.14. Found: C, 52.32; H, 4.57; N,

10.26.

2-[3-[4-Fluoro-3-(trifluoromethyl)phenyl]ureido]-2-methylpropanoic acid (AR37).

Hydantoin AH01 (1.20 g, 4.1 mmol) was added to 2 M NaOH (40 mL). The reaction mixture was stirred at rt for 4 h, and then quenched with 2 M HCl (50 mL). The precipitate was filtered and rinsed by H2O (20 mL) and then dried to afford AR37 as a white solid (1.20 g, 95%). mp 196.5–197.5 °C;

1 H NMR (DMSO-d6) δ 1.43 (s, 6H), 6.61 (s, 1H), 7.37 (t, J = 9.8 Hz, 1H), 7.47-7.50

(m,1H), 7.96-7.97 (m, 1H), 8.87 (s, 1H), 12.44 (s, 1H);

13 C NMR (DMSO-d6) δ 25.42, 55.13, 115.12 (d, J = 5.0 Hz), 116.45 (qd, J = 31.6, 12.8

Hz), 117.65 (d, J = 21.5 Hz), 122.85 (q, J = 272.1 Hz), 123.42 (d, J = 7.8 Hz), 137.34,

153.32 (d, J = 246.9 Hz), 154.37, 176.31.

Anal. Calcd for C12H12F4N2O3: C, 46.76; H, 3.92; N, 9.09. Found: C, 46.50; H, 4.04;

N, 9.19

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5-(hydroxymethyl)-5-methylimidazolidine-

2,4-dione (AR40)

Step 1.To a solution of 2-amino-3-hydroxy-2-methylpropanoic acid (537 mg, 4.5 mmol) in dioxane (23 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (8.10 g,

39.5) under Ar. After the reaction mixture was stirred at 80 oC for 6 h, it was cooled to rt, 76

diluted with CH2Cl2 (25 mL) and extracted with 2 M NaOH (3 × 25 mL). After the combined aq. layers were separated and filtered to remove the insoluble N,N-diarylurea side product, the filtrate was acidified with 2 M HCl (100 mL) and concentrated. Step 2.

The residue was suspended in 2 M HCl (50 mL) and then heated at 110°C for 4 h. After the reaction mixture was cooled to rt, the resulting precipitate was collected by filtration to give AR40 (370 mg, 27%) as a white solid. mp 166–168 °C;

1 H NMR (DMSO-d6) δ 1.30 (s, 3H), 3.43 (d, J = 10.7 Hz, 1H), 3.67 (d, J = 11.2 Hz, 1H),

7.66 (t, J = 10.3 Hz, 1H), 7.76-7.77 (m, 1H), 7.81 (d, J = 6.3, 1H), 8.52 (s, 1H);

13 C NMR (DMSO-d6) 18.97, 63.52, 65.38, 117.00 (q, J = 32.5 Hz), 118.07 (d, J = 22.0

Hz), 122.40 (q, J = 273.0 Hz), 125.20, 129.15, 133.29 (d, J = 9.2 Hz), 154.78, 157.61 (d,

J = 254.2 Hz), 175.04.

Anal. Calcd for C12H10F4N2O3: C, 47.07; H, 3.29; N, 9.15. Found: C, 46.91, H, 3.46, N,

9.32

5,5-Dimethyl-3-[2-(trifluoromethyl)pyridin-4-yl]imidazolidine-2,4-dione (AR43)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (776 mg, 6.1 mmol) and Cu2O (288 mg, 2.0 mmol) in DMA (30 mL) was added 4-iodo-2-(trifluoromethyl)pyridine (750 mg,

2.8 mmol). After stirring at 140 oC for 24 h, the solvent was evaporated in vacuo to give a crude product which was further purified by sg chromatography using successive elution with hexane and EtOAc to afford AR43 as a white solid (674 mg, 90%). mp 195–197 oC; 77

1 H NMR (DMSO-d6) δ 1.43 (s, 6H), 7.95 (dd, J = 5.4, 1.9 Hz, 1H), 8.14 (d, J = 1.8 Hz,

1H), 8.87 (d, J=5.4 Hz, 1H), 8.89 (br s, 1H);

13 C NMR (DMSO-d6) δ 24.61, 57.82, 116.17, 121.39 (q, J = 274.3 Hz), 122.29, 141.72,

146.94 (q, J = 34.1 Hz), 151.15, 152.68, 175.71.

Anal Calcd for C11H10F3N3O2: C, 48.36; H, 3.69; N, 15.38. Found: C, 47.97; H, 4.00; N,

15.64.GC-MS purity: 100%

5,5-Dimethyl-3-(pyrazin-2-yl)imidazolidine-2,4-dione (AR48)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (1.28 g, 10.0 mmol) and Cu2O

(357.5 mg, 2.5 mmol) in DMA (20 mL) was added 2-iodopyrazine (1.00 g, 4.9 mmol).

The reaction mixture was then heated to 140 oC for 24 h. After solvent removal in vacuo, the crude product was purified by sg chromatography using successive elutions with hexane, EtOAc, and ethanol. After removal of the solvents in vacuo, the residue was crystallized from a mixture of ether (0.5 mL) and hexane (10 mL). The resulting precipitate was collected by filtration to give A48 as a pale yellow solid (210 mg, 21%). mp 149-151 oC;

1 H NMR (DMSO-d6) δ 1.44 (s, 6H), 8.66-8.90 (m, 4H);

13 C NMR (DMSO-d6) δ 24.65, 58.38, 142.76, 143.72, 143.91, 144.38, 153.05, 175.80.

Anal Calcd for C9H10N4O2; C, 52.42; H, 4.89; N, 27.17. Found: C, 52.34; H, 5.00;

N, 27.21. 78

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethyldihydropyrimidine-2,4(1H,3H)- dione (AR51)

Step 1. To a solution of 3-amino-2,2-dimethylpropanoic acid hydrochloride (700 mg, 4.6 mmol) in 2 M NaOH (5 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene

(1.95 g, 9.5 mmol), and the reaction mixture was stirred at rt for 4 h. After filtration to remove the insoluble N,N-diarylurea side product, the filtrate was adjusted to pH=1.0 with 2 M HCl and concentrated to afford 3-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]-

2,2-dimethylpropanoic acid (480 mg, 32%) as a white solid.

1 H NMR (DMSO-d6) δ 1.09 (s, 6H), 3.22 (d, J = 6.2 Hz, 2H), 6.25 (t, J = 6.2 Hz, 1H), 7.36

(t, J = 9.8 Hz, 1H), 7.43-7.53 (m , 1H), 7.95 (dd, J = 6.5, 2.8 Hz, 1H), 8.99 (s, 1H), 12.39

(s, 1H).

Step 2. 3-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]-2,2-dimethylpropanoic acid (450 mg, 1.4 mmol) was suspended in 2 M HCl (50 mL) and then heated at 110 °C for 12 h.

After the reaction mixture was cooled to rt, the resulting precipitate was filtered and washed with saturated NaHCO3 (30 mL) to give AR51 as a white solid (355 mg, 83%). mp 214–215 oC;

1 H NMR (DMSO-d6) δ 1.21 (s, 6H), 3.18 (d, J= 2.8 Hz, 2H), 7.52-7.59 (m, 2H), 7.63-7.69

(m, 1H), 8.11 (s, 1H); 79

13 C NMR (DMSO-d6) δ 22.35, 37.39, 46.31, 116.60 (qd, J = 32.9, 13.7 Hz), 117.41 (d, J

= 21.5 Hz), 122.34 (q, J = 271.9 Hz), 128.34, 133.07 (d, J = 3.5 Hz), 136.42 (d, J = 9.2

Hz), 153.02, 157.88 (d, J = 253.6 Hz), 175.33.

Anal Calcd for C13H12F4N2O2: C, 51.32; H, 3.98; N, 9.21. Found: C, 51.19; H, 3.99; N,

9.12.

5,5-Dimethyl-3-[6-(trifluoromethyl)pyridin-3-yl]imidazolidine-2,4-dione (AR52)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (384 mg, 3.00 mmol) and Cu2O (143 mg, 1.00 mmol) in DMA (10 mL) was added 5-iodo-2-(trifluoromethyl)pyridine (476 mg,

1.74 mmol), and the mixture was heated to 140 oC for 48 h. After solvent removal in vacuo, the crude product was purified by sg chromatography using successive elution with hexane and EtOAc. After removal of the solvents in vacuo, the residue was crystallized from 20:1 hexane and EtOAc. The resulting precipitate was collected by filtration and rinsed with H2O (5 mL) to give AR52 as a white solid (357 mg, 75%). mp 173–174 °C;

1 H NMR (DMSO-d6) δ 1.44 (s, 6 H), 8.08 (d, J=8.3 Hz, 1 H), 8.20 (d, J=8.3 Hz, 1 H), 8.80

(s, 1 H), 8.88 (s, 1 H);

13 C NMR (DMSO-d6) δ 24.80, 58.36, 121.16, 121.61(q, J = 273.9 Hz), 132.27, 135.50,

144.63 (q, J = 34.4 Hz), 147.43, 153.32,176.10.

Anal. Calcd forC11H10F3N3O2: C, 48.36; H, 3.69; N, 15.38. Found C, 48.48; H, 3.81; N,

15.16. 80

5,5-Dimethyl-3-(pyridin-4-yl)imidazolidine-2,4-dione (AR53)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (5.38 g, 42 mmol) and Cu(OAc)2

(543 mg, 3mmol) in methanol (150 mL) was added pyridin-4-ylboronic acid (3.6 g, 29

o mmol) under O2.The mixture was heated at 70 C overnight. The solvent was then filtered through silica gel to remove copper and the resulting cake was washed with methanol (100 mL). Evaporation of the filtrate gave a crude product which was further purified by crystallization from a saturated Na2CO3 solution (20 mL) to afford AR53 as a white solid (2.8 g, 47%). mp 201–203 oC;

1 H NMR (DMSO-d6) δ 1.42 (s, 6H), 7.57 (d, J = 5.6 Hz, 2H), 8.67 (d, J = 6.1 Hz, 2H),

8.74 (brs, 1H).

13 C NMR (DMSO-d6) δ 24.70, 57.75, 119.62, 139.85, 150.26, 153.08, 175.88.

Anal Calcd for C10H11N3O2: C, 58.53; H, 5.40; N, 20.48. Found: C, 58.14; H, 5.64; N,

20.57.

5-(4,4-Dimethyl-2,5-dioxoimidazolidin-1-yl)-2-fluoro-N,N-dimethylbenzamide

(AR54) 81

To a solution of 5,5-dimethylimidazolidine-2,4-dione (640 mg, 5.0mmol) and Cu2O (259 mg, 1.8 mmol) in DMA (10 mL) was added 2-fluoro-5-iodo-N,N-dimethylbenzamide (496 mg, 1.7 mmol). The mixture was then heated to 150 oC for 48 h. Solvent removal in vacuo gave a crude product which was further purified by sg chromatography using successive elution with hexane, EtOAc and ethanol followed by crystallization from a mixture EtOAc:hexane (1:10) to afford AR54 as a white solid (296 mg, 58%). mp 166–168 oC;

1 H NMR (DMSO-d6) δ 1.40 (s, 6H), 2.87 (s, 3H), 3.01 (s, 3H), 7.41 (t, J = 9.1 Hz, 1H),

7.44 (dd, J = 6.0, 2.5 Hz, 1H), 7.51 (ddd, J = 8.9, 4.8, 2.6 Hz, 1H), 8.59 (s, 1H).

13 C NMR (DMSO-d6) δ 24.68, 34.33, 37.85, 57.85, 116.21 (d, J = 23.3 Hz), 124.76 (d, J

= 20.0 Hz), 127.11 (d, J = 4.7 Hz), 128.69 (d, J = 3.0 Hz), 129.62 (d, J = 8.7 Hz), 153.93,

156.32 (d, J = 246.5 Hz), 164.45, 176.27.

Anal Calcd for C14H16FN3O3: C, 57.33; H, 5.50; N, 14.33. Found: C, 57.51; H, 5.25; N,

14.22.

5,5-Dimethyl-3-(quinolin-4-yl)imidazolidine-2,4-dione (AR55)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (384 mg, 3 mmol) and Cu2O (142 mg, 1 mmol) in DMA (5 mL) was added 4-iodoquinoline (163 mg, 0.64 mmol). After the reaction mixture was heated at 150 oC for 72 h, the solvent was removed in vacuo. The residue was purified by sg chromatography using successive elution with hexane, EtOAc and ethanol to afford AR55 as a pale yellow solid (101 mg, 62%). mp 194–196 oC;

1 H NMR (DMSO-d6) δ 1.50 (s, 3H), 1.57 (s, 3H), 7.61 (d, J = 4.6 Hz, 1H), 7.65-7.76 (m,

2H), 7.86 (t, J = 7.2 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.83 (s, 1H), 9.05 (d, J = 4.6 Hz,

1H);

13 C NMR (DMSO-d6) δ 24.50, 25.26, 58.77, 121.60, 122.83, 124.72, 127.58, 129.42,

130.15, 137.08, 148.77, 150.73, 153.60, 176.45.

Anal Calcd for C14H13N3O2·H2O: C, 61.53; H, 5.53; N, 15.38. Found: C, 61.82; H, 5.08;

N, 15.30.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethylpyrrolidine-2,4-dione (AR56)

Step 1. A mixture of 2-[4-fluoro-3-(trifluoromethyl)phenyl]acetic acid (2.22 g, 10 mmol)

o and SOCl2 (4 mL) was stirred at 80 C overnight. The reaction mixture was concentrated in vacuo to afford 2-[4-fluoro-3-(trifluoromethyl)phenyl]acetyl chloride which was used for the next step without further purification. Step 2. To a solution of methyl 2-amino-2- methylpropanoate HCl salt (1.53 g, 10 mmol) and Et3N (5.0 g, 50 mmol) in THF (47.5 mL) was added acetyl chloride in THF (2.5 mL), and the reaction mixture was stirred at rt 83

overnight. After filtration to remove insoluble TEA HCl salt, the filtrate was concentrated in vacuo. The residue was purified by chromatography (sg, EtOAc:hexane, 1:1) to afford methyl 2-[2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido]-2-methylpropanoate as a white

1 solid (1.13 g, 35%). H NMR (DMSO-d6) δ 1.35 (s, 6H), 3.50 (s, 3H), 3.52 (s, 2H), 7.45

(dd, J = 10.9, 8.6 Hz, 1H), 7.52-7.61 (m, 1H), 7.63 (d, J = 7.7 Hz, 1H), 8.54 (s, 1H). Step

3. To a solution of methyl 2-[2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido]-2- methylpropanoate (860 mg, 2.68 mmol) in THF (7 mL) was added a suspension of NaH

(300 mg, 12.5 mmol) in THF (6.4 mL) dropwise under Ar. The reaction mixture was stirred at rt for 12 h before quenching with a mixture of acetic acid (1.2 g, 20 mmol) and

H2O (1 mL). Solvent removal in vacuo gave a crude product which was crystallized from hexane to afford AR56 as a white solid (406 mg, 52%). mp 217–219 oC;

1 H NMR (DMSO-d6) δ 1.36 (s, 6H), 7.46 (dd, J = 10.8, 8.9 Hz, 1H), 7.83 (s, 1H), 8.30-

8.40 (m, 1H), 8.47 (dd, J = 7.4, 2.2 Hz, 1H), 11.68 (s, 1H).

13 C NMR (DMSO-d6) δ 24.54, 56.49, 98.64, 115.88 (qd, J = 31.8, 12.2 Hz), 116.56 (d, J

= 20.1 Hz), 122.87 (q, J = 271.4 Hz), 124.55 (q, J = 4.8 Hz), 129.75 (d, J = 3.7 Hz),

132.87 (d, J = 8.0 Hz), 156.37 (d, J = 250.9 Hz), 170.39, 176.13.

Anal Calcd for C13H11F4NO2: C, 53.99; H, 3.83; N, 4.84. Found: C, 54.09; H, 3.92; N,

4.60.

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethyl-1-

(methylsulfonyl)imidazolidine-2,4-dione (AR57) 84

To a solution of AH01 (700 mg, 2.4mmol) in THF (7.5 mL) was added NaH (100 mg, 4.1 mmol) in THF (7.5 mL) at 0 oC under Ar. The reaction mixture was then stirred at rt for

30 min before dropwise addition of methanesulfonyl chloride (410 mg, 3.6mmol). After further stirring at rt for 72 h, the reaction was quenched with acetic acid (4 mL). Removal of solvents in vacuo gave a residue to which was added H2O (30 mL). The resulting precipitate was filtered and crystallized from EtOAc:hexane (1:5) to afford AR57 as a white solid (812 mg, 92%). mp 207–208 oC;

1 H NMR (CDCl3) δ 1.86 (s, 6H), 3.44 (s, 3H), 7.35 (t, J = 9.2 Hz, 1H), 7.67 (dt, J = 7.4,

3.5 Hz, 1H), 7.75 (dd, J = 6.1, 2.7 Hz, 1H).

13 C NMR (CDCl3) δ 24.19, 43.53, 66.73, 118.19 (d, J = 22.3 Hz), 119.70 (qd, J = 33.9,

13.9 Hz), 121.95 (q, J = 272.6 Hz), 125.36 (qd, J = 6.9, 4.5 Hz), 126.56, 131.70 (d, J =

9.4 Hz), 151.70, 159.31 (d, J = 259.9 Hz), 173.02.

Anal Calcd for C13H12F4N2O4S: C, 42.39; H, 3.28; N, 7.61. Found: C, 42.40; H, 3.46; N,

7.49.

1-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethylimidazolidine-2,4-dione (AR58) 85

Step 1. To a mixture of 4-fluoro-3-(trifluoromethyl)aniline (1.791 g, 10 mmol), formamide

o (50 mL) and acetone (25 mL) was added 1 M TiCl4 in CH2Cl2 (12.5 mL) at 0 C under Ar.

After 1 h, Zn powder (1.5 g, 23 mmol) was added followed by dropwise addition of 50%

H2O2 (1.75 mL) in formamide (23.25 mL) over 3 h. The reaction was quenched with H2O

(25 mL) and adjusted to pH=8 with conc. ammonium hydroxide followed by extraction with EtOAc (3 × 50 mL). The combined organic layers were concentrated in vacuo and the residue was dissolved in CHCl3 (100 mL). The solution was then washed with H2O (3

× 25 mL). Evaporation gave a crude product which was further purified by successive recrystallizations from a mixture of CH2Cl2 (5 mL) and hexane (1 mL) and a mixture of acetic acid (150 mg) and H2O (1 mL) to afford 2-[[4-fluoro-3-

(trifluoromethyl)phenyl]amino]-2-methylpropanamide as a white solid (418 mg, 16%); 1H

NMR (DMSO-d6) δ 1.35 (s, 6H), 6.18 (s, 1H), 6.71 (dt, J = 9.0, 3.6 Hz, 1H), 6.81 (dd, J =

13 6.0, 3.0 Hz, 1H), 7.05 (s, 1H), 7.22 (t, J = 9.8 Hz, 1H), 7.34 (s, 1H); C NMR (DMSO-d6)

δ 25.22, 56.63, 111.09 (q, J = 4.7 Hz), 116.20 (qd, J = 31.5, 13.0 Hz), 117.16 (d, J =

21.2 Hz), 118.79 (d, J = 7.4 Hz), 122.92 (q, J = 272.0 Hz), 143.20 (d, J = 1.9 Hz), 150.44

(dq, J = 241.0, 2.4 Hz), 177.13. Step 2. To a solution of 2-[[4-fluoro-3-

(trifluoromethyl)phenyl]amino]-2-methylpropanamide (160 mg, 0.6 mmol) in toluene (5 mL) was added 2-isocyanato-1,3-diisopropylbenzene (150 µL) using a pipette. After the mixture was heated to 250 oC at 5 bar for 10 min under microwave irradiation in a sealed tube, the solvent was evaporated in vacuo. The residue was purified by sg chromatography eluting successively with CH2Cl2 and acetone:hexane (1:3) to afford

AR58 as a white solid (108 mg, 62%). 86

mp 193–195 oC;

1 H NMR (DMSO-d6) δ 1.34 (s, 6H), 7.63 (t, J = 9.7 Hz, 1H), 7.71–7.78 (m, 1H), 7.84 (dd,

J = 6.6, 2.6 Hz, 1H), 11.26 (s, 1H);

13 C NMR (DMSO-d6) δ 23.09, 64.21, 117.25 (qd, J = 32.8, 13.2 Hz), 118.25 (d, J = 21.5

Hz), 122.18 (q, J = 273.5 Hz), 128.19, 131.44 (d, J = 3.4 Hz), 135.86 (d, J = 9.2 Hz),

154.99, 157.87 (d, J = 254.8 Hz), 177.14.

Anal Calcd for C12H10F4N2O2: C, 49.66; H, 3.47; N, 9.65. Found: C, 49.71, H, 3.58, N,

9.80

3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5-methylimidazolidine-2,4-dione (AR19B)

Step 1. To a solution of 2-aminopropanoic acid (2.225 g, 25 mmol) in 1 M NaOH (40 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (6.15 g, 30 mmol) in CH3CN

(15 mL) at 0 oC dropwise. The reaction mixture was stirred at 0 oC for 3 h and then warmed to rt overnight. Step 2. The reaction mixture was adjusted to pH=3.0 by 32%

HCl and concentrated in vacuo. The resulting precipitate was crystallized from

EtOAc:hexane (1:40) to give 2-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]propanoic acid as a white solid (5.157 g, 70%), 1.1 g (3.74 mmol) of which was suspended in 4 M

HCl (50 mL) and then heated at 110 °C overnight. After the reaction mixture was cooled to rt, the resulting precipitate was filtered to afford AR19B as a white solid (845 mg,

82%). mp 153–154 oC; 87

1 H NMR (DMSO-d6) δ 1.38 (d, J = 6.9 Hz, 3H), 4.27 (q, J = 6.9 Hz, 1H), 7.65 (t, J = 9.7

Hz, 1H), 7.76 – 7.83 (m, 1H), 7.87 (dd, J = 6.7, 2.5 Hz, 1H), 8.59 (s, 1H);

13 C NMR (DMSO-d6) δ 16.94, 52.23, 116.73 (qd, J = 32.8, 13.4 Hz), 117.76 (d, J = 21.8

Hz), 122.23 (q, J = 272.1 Hz), 125.47 (q, J = 5.1 Hz), 128.95 (d, J = 3.5 Hz), 133.43 (d, J

= 9.3 Hz), 154.84, 157.50 (dq, J = 254.3, 2.1 Hz), 173.81.

Anal Calcd for C11H8F4N2O2·0.5H2O: C, 46.33; H, 3.18; N, 9.82; Found: C, 46.61; H, 3.19;

N, 9.81.

3-[3-(Difluoromethyl)-4-fluorophenyl]-5,5-dimethylimidazolidine-2,4-dione (AR64)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (2.54 g, 19.8 mmol) and Cu2O (1.09 g, 7.6 mmol) in DMA (30 mL) was added 4-bromo-2-(difluoromethyl)-1-fluorobenzene

(3.50 g, 15.5 mmol). After the reaction mixture was heated at 160 oC for 24 h, the solvent was removed in vacuo. The residue was purified by chromatography (sg, EtOAc) and then crystallized successively from hexane (15 mL) and H2O (20 mL) to afford AR64 as a white solid (3.43 g, 81%). mp 158–159oC;

1 H NMR (DMSO-d6) δ 1.41 (s, 6H), 7.26 (t, J = 54.1 Hz, 1H), 7.51 (t, J = 9.5 Hz, 1H),

7.59 – 7.68 (m, 1H), 7.72 (dd, J = 6.4, 2.5 Hz, 1H), 8.62 (s, 1H). 88

13 C NMR (DMSO-d6) δ 24.60, 57.85, 111.52 (td, J = 236.2, 3.5 Hz), 116.72 (d, J = 21.8

Hz), 121.46 (td, J = 23.4, 14.0 Hz), 125.75 (d, J = 3.3 Hz), 128.68 (d, J = 3.3 Hz), 131.64

(d, J = 9.0 Hz), 153.79, 158.38 (dt, J = 251.3, 4.8 Hz), 176.17.

Anal Calcd for C12H11F3N2O2: C, 52.94; H, 4.07; N, 10.29. Found: C, 52.72, H, 4.19, N,

10.12.

2-[3-[4-Fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethyl-2,4-dioxoimidazolidin-1- yl]acetamide (AR63)

To a solution of AH01 (1.04 g, 3.6 mmol) in THF (15 mL) was added NaH (163 mg, 6.8 mmol) in THF (15 mL) at 0 oC under Ar. The mixture was then stirred at rt for 3 h before dropwise addition of 2-bromoacetamide (937 mg, 6.8 mmol). The reaction mixture was stirred at rt for 72 h before quenching with acetic acid (600 mg, 10 mmol). Solvent removal in vacuo gave a residue which was crystallized from CH2Cl2 to afford AR63 as a white solid (600 mg, 48%). mp 186–187 oC;

1 H NMR (DMSO-d6) δ 1.43 (s, 6H), 3.90 (s, 2H), 7.25 (s, 1H), 7.50 (s, 1H), 7.69 (t, J =

9.7 Hz, 1H), 7.80-7.86 (m, 1H), 7.90 (dd, J = 6.6, 2.5 Hz, 1H); 89

13 C NMR (DMSO-d6) δ 22.04, 41.90, 61.62, 116.72 (qd, J = 32.9, 13.6 Hz), 117.84 (d, J

= 21.8 Hz), 122.21 (q, J = 272.4 Hz), 125.36 (q, J = 5.0 Hz), 128.74 (d, J = 3.3 Hz),

133.37 (d, J = 9.3 Hz), 153.50, 157.56 (dq, J = 254.9, 1.9 Hz), 169.63, 175.02.

Anal Calcd for C14H13F4N3O3: C, 48.42; H, 3.77; N, 12.10. Found: C, 48.70, H, 4.01, N,

11.89

1-Ethyl-3-[4-fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethylimidazolidine-2,4-dione

(AR62)

To a solution of AH01 (700 mg, 2.4 mmol) in THF (7.5 mL) was added NaH (87 mg, 3.6 mmol) in THF (7.5 mL) at 0 oC under Ar. The reaction mixture was then stirred at rt for 3 h before dropwise addition of iodoethane (561 mg, 3.6 mmol). The reaction mixture was stirred at rt for 72 h before quenching with acetic acid (600 mg, 10 mmol) and H2O (1 mL). Solvent removal in vacuo gave a residue which was crystallized from H2O. The residue was further purified by chromatography (sg, EtOAc:hexane, 1:4) to afford AR62 as a white solid (301 mg, 39%). mp 90–91 oC;

1 H NMR (CDCl3) δ 1.32 (t, J = 7.2 Hz, 3H), 1.53 (s, 6H), 3.43 (q, J = 7.2 Hz, 2H), 7.29 (t,

J = 9.4 Hz, 1H), 7.65-7.74 (m, 1H), 7.78 (dd, J = 6.3, 2.6 Hz, 1H). 90

13 C NMR (CDCl3) δ 15.14, 23.58, 35.08, 61.97, 117.60 (d, J = 22.1 Hz), 119.11 (qd, J =

33.6, 13.6 Hz), 122.21 (q, J = 272.5 Hz), 124.95 (q, J = 4.5 Hz), 128.25 (d, J = 3.6 Hz),

131.34 (d, J = 8.9 Hz), 153.46, 158.60 (d, J = 258.1 Hz), 175.28.

Anal Calcd for C14H14F4N2O2: C, 52.83; H, 4.43; N, 8.80. Found: C, 52.80, H, 4.60, N,

8.71

5,5-Dimethyl-3-[4-methyl-3-(trifluoromethyl)phenyl]imidazolidine-2,4-dione (AR59)

To a solution of 5,5-dimethylimidazolidine-2,4-dione (568 mg, 4.44 mmol) and Cu2O (240 mg, 1.68mmol) in DMA (15 mL) was added 4-iodo-1-methyl-2-(trifluoromethyl)benzene

(1.00 g, 3.5 mmol). After stirring at 160 oC for 12 h, the solvent was evaporated in vacuo to give a crude product which was further purified by sg chromatography using successive elution with hexane, EtOAc, and EtOH to afford AR59 as a white solid (536 mg, 54%). mp 134–135 oC;

1 H NMR (CDCl3) δ 1.57 (s, 6H), 2.51 (s, 3H), 5.52 (s, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.50

(d, J = 7.9 Hz, 1H), 7.70 (s, 1H);

13 C NMR (DMSO-d6) δ 18.43, 24.65, 57.85, 123.92 (q, J = 5.2 Hz), 124.09 (q, J = 273.6

Hz), 127.54 (q, J = 29.6 Hz), 130.37, 130.41, 132.59, 135.53, 153.85, 176.21.

Anal Calcd for C13H13F3N2O2: C, 54.55; H, 4.58; N, 9.79; Found: C, 54.45; H, 4.73; N,

9.62. 91

3.6.2. Antischistosomal Screen.

As described by Keiser (2010), cercariae of S. mansoni were obtained from infected

Biomphalaria glabrata. NMRI mice were infected subcutaneously with approximately 100

S. mansoni cercariae. At 49 d after infection, groups of four mice were treated with single 100 mg/kg oral doses of compounds in a 7% (v/v) Tween 80% and 3% (v/v) ethanol vehicle (10 mL/kg). Untreated mice (n = 8) served as controls. At 21 d post- treatment, animals were killed by the CO2 method and dissected. Worms were removed by picking, then sexed and counted.

3.6.3. Androgen-Dependent Cell-based Assay.

As described by Jones and Diamond (2008), transfected LAPC4 cells were cultured in phenolredfree RPMI 1640 media supplemented with antibiotics and 10% FBS. Cells were then exposed to 10 nM DHT for 24 h in the presence of varying concentrations of test compounds. Control wells on eachplate included no DHT (base line signal) and DHT without test compounds (maximal signal). After 24 h, normalized luciferase signals were measured. Nilutamide (N) was used as a positive control. Mean-effect plots from four independent experiments were generated to determine IC50 values for the test compounds.

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Chapter 4.

Urea carboxylic acid antischistosomal agents

Abstract

Urea carboxylic acid AR37, the product formed by hydrolysis of the hydantoin substructure of Ro 13-39781, has substantial in vivo antischistosomal efficacy. Base on this finding, several efficient synthetic routes were developed to make the first round of urea carboxylic acid targets. These methods can be applied to the synthesis of structurally diverse AR37 analogs. These urea carboxylic acids could lead to an advance in the structure–activity relationship of this class of compounds in antischistosomal researches.

4.1 Introduction

Schistosomiasis is a tropical parasitic disease2 caused by infections with flukes of the genus Schistosoma. Of these, Schistosoma mansoni, S. haematobium, and S. japonicum cause the largest public health burden3-5. Praziquantel (PZQ) is the only drug available for treatment of this disease6, 7. The high drug pressure from the widespread administration of PZQ could lead to problematic drug resistance8, 9. Even so, drug discovery for schistosomiasis has languished10, 11.

In Chapter 3, I discovered that urea carboxylic acid AR37, the product formed by hydrolysis of the hydantoin substructure of Ro 13-39781, has substantial in vivo antischistosomal efficacy. Further, investigations by Roche scientists1 revealed that the methyl ester of AR37 had a single oral dose ED50 of 62 mg/kg. No other analogs of

AR37 were investigated for antischistosomal activity.

4.2 Design

Previously, we showed that a single 100 mg/kg oral dose of AR37 administered to S. mansoni-infected mice reduced adult WBR by 67%. However, like Ro 13-3978,

AR37 at concentrations up to 100 μg/mL (325 μM) had no measurable effect on ex vivo

S. mansoni. We also found that AR37 at concentrations up to 30 μM was not cytotoxic to rat skeletal myoblast L6 cells. Thus, we suggest that the relatively high antischistosomal efficacy, low cytotoxicity, and good physicochemical properties (Log D of 3.1 and PSA of

78 Å) of AR37 indicate that this urea carboxylic acid is an attractive starting point for optimization.

In this respect, we designed the first round of urea carboxylic acid targets. They include the primary amide U01, the unsubstituted analog U06, the conformationally- restricted UC04, the succinamic acid UC05 and the phenylacetamide derivative UC06.

In UC03, UC07 and UC08, the gem-dimethyl group of AR37 is replaced with the H

(UC03), cyclopropyl (UC07) and cyclobutyl (UC08) groups.

4.3 Chemistry

U06, UC03, UC07, and UC08 were obtained by reaction of phenyl isocyanates

(1a, 1b) with α-amino acids (2a, 2b, 2c, 2d) in aq. NaOH to form the corresponding urea carboxylic acids (Scheme 4.1.). Conformationally-restricted urea UC04 (cyclouracil) was obtained by exposure of the Ro 13-3978 with 1-bromo-3-chloropropane according to the method of Kawamura et al12 (Scheme 4.2.). U01 was obtained by reaction of conc.

NH4OH with activated AR37 (Scheme 4.2.). Succinamic acid UC05 was obtained by acylation of 4-fluoro-3-trifluoromethylaniline with 2,2-dimethylsuccinic anhydride according to the published method13 (Scheme 4.3.). UC06, the phenylacetamide derivative of AR37, was obtained by the acylation of 2-amino-2-methyl propanoic acid with corresponding acid chloride according to the method of Buijs et al14 (Scheme 4.4.). 97

Scheme 4.1.

(a) 1-2 M NaOH, 0 oC to rt, 12 h

Scheme 4.2.

(a) 2 M NaOH, rt, 4 h;

(b) HOBt, EDCI·HCl, DMA, rt, 12 h;

(d) NaH, DMF, rt, 2 h, then 1-bromo-3-chloropropane, rt, 48 h.

Scheme 4.3.

(a) THF, rt, 12 h

Scheme 4.4.

(a) 2 M NaOH, 0 oC, 2 h

100

Scheme 4.5.

(a) SOCl2, DMF, CH2Cl2, rt, 12 h;

(b) 4-fluoro-3-(trifluoromethyl)aniline, DMA, rt, 12 h;

101

4.4. Discussion

4.4.1. Synthesis of U04

The proposed mechanism of the formation of UC04 was shown in Figure 4.1.

The first step was an intermolecular N-alkylation of the hydantoin AR02 to form N1-3- chloropropyl hydantoin (3a). This hydantoin 3a was further hydrolyzed and then intramolecularly alkylated under the basic condition to form UC04. The trace amount of

NaOH resulted from the hydrolysis of NaH.

4.4.2. Synthesis of U05

Two different approaches were explored for the synthesis of succinamic acid

UC05. One of these approaches was to hydrolyze methyl ester 4a by NaOH to produce

UC05. However, 1H NMR showed an 1:1 ratio of UC05 and UC09 (Figure 4.2.). They were formed by the cyclization of methyl ester 4a and then hydrolysis under basic condition. Another approach included acylation of 4-fluoro-3-trifluoromethylaniline with

2,2-dimethylsuccinic anhydride according to the published method.13 The 1H NMR showed a 95% selectivity for UC05 (Figure 4.3.). The crude was crystallized from CH2Cl2

(10 mL) to afford UC05 (93%) as a white solid.

4.5 Summary

Several efficient synthetic routes were developed to make the first round of urea carboxylic acid targets. These methods can be applied to the synthesis of structurally diverse AR37 analogs. These urea carboxylic acids could lead to an advance in the structure–activity relationship of this class of compounds in antischistosomal researches.

102

Figure 4.1. Proposed mechanism for the formation of UC04

103

Figure 4.2. 1H NMR of UC05 and UC09 (1:1)

104

Figure 4.3. 1H NMR of UC05 and UC09 (20:1)

105

4.6 Experimental

General.

Melting points are uncorrected. 1D 1H and 13C NMR spectra were recorded on a

500 MHz spectrometer using CDCl3 or DMSO-d6 as solvents. All chemical shifts are

1 reported in parts per million (ppm) and are relative to internal (CH3)4Si (0 ppm) for H

13 and CDCl3 (77.2 ppm) or DMSO-d6 (39.5 ppm) for C NMR. EI GC-MS data were obtained using a quadrapole mass spectrometer with 30 m DB-XLB type columns and a

He flow rate of 1 mL/min. Silica gel (sg) particle size 32−63 μm was used for all flash column chromatography. Reported reaction temperatures are those of the oil bath.

Synthesis of U01.

3-[4-fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethylimidazolidine-2,4-dione (1.20 g,

4.1 mmol) was added 2 M NaOH (40 mL). After it was stirred at rt for 4 h, the reaction was quenched and adjusted to pH=3.0 with 2 M HCl. The resulting precipitate was collected by filtration to give 2-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]-2- methylpropanoic acid as a white solid (1.20 g, 95%).

1 H NMR (500 MHz, DMSO-d6) δ 1.42 (s, 6H), 6.60 (s, 1H), 7.36 (t, J = 9.8 Hz,

1H), 7.42 – 7.55 (m, 1H), 7.96 (dd, J = 6.5, 2.8 Hz, 1H), 8.87 (s, 1H), 12.43 (s, 1H).

To a solution of 2-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]-2-methylpropanoic acid(1.2 g, 3.9 mmol) and N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride

(1.1 g, 5.7 mmol) in DMA (60 mL) was added N-Hydroxy succinimide (552 mg, 4.8 mmol). After it had been stirred at rt for overnight, the reaction was quenched with H2O

(300 mL). The resulting precipitate was collected by filtration to give 2,5-dioxopyrrolidin- 106

1-yl 2-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]-2-methylpropanoate (560 mg, 35%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 1.57 (s, 6H), 2.79 (bs, 4H), 7.07 (s, 1H), 7.38 (t,

J = 9.7 Hz, 1H), 7.52–7.58 (m, 1H), 7.96 (d, J = 6.1 Hz, 1H), 8.83 (s, 1H).

The mixture of 2,5-dioxopyrrolidin-1-yl 2-[3-[4-fluoro-3-

(trifluoromethyl)phenyl]ureido]-2-methylpropanoate (560 mg, 1.38 mmol) in 28% wt ammonium hydroxide (20 mL) was stirred at rt for 1.5 h. The precipitation was collected by filtration and washed with 2 M NaOH (10 mL) to afford 2-[3-[4-fluoro-3-

(trifluoromethyl)phenyl]ureido]-2-methylpropanamideas a white solid (350 mg, 83%)

1 H NMR (500 MHz, DMSO-d6) δ 1.45 (s, 6H), 6.60 (s, 1H), 7.06 (s, 1H), 7.32 (s,

1H), 7.35 (t, J = 9.8 Hz, 1H), 7.41–7.51 (m, 1H), 7.97 (dd, J = 6.6, 2.7 Hz, 1H), 9.10 (s,

1H).

13 C NMR (125 MHz, DMSO-d6) δ 25.05, 55.53, 114.71 (d, J = 4.3 Hz), 116.20 (qd,

J = 31.8, 12.8 Hz), 117.41 (d, J = 21.3 Hz), 122.68 (q, J = 272.5 Hz), 123.00 (d, J = 7.8

Hz), 137.46 (d, J = 2.6 Hz), 152.96 (d, J = 246.8 Hz), 153.78, 176.81.

mp 205–206 oC

Anal Calcd for C12H13F4N3O2: C, 46.91; H, 4.26; N, 13.68; Found: C, 46.82; H,

4.32; N, 13.35

Synthesis of U06.

107

To a solution of 2-amino-2-methylpropanoic acid (1.03 g, 10 mmol) in 2 M NaOH

(10 mL) was added isocyanatobenzene (2.38 g, 20 mmol) at rt. The mixture was stirred at rt overnight. The insoluble diaryl urea side product in the reaction mixture was removed by filtration. The filtrate was adjusted to pH=3.0 with 2 M HCl. The resulting precipitate was collected by filtration to give 2-methyl-2-(3-phenylureido)propanoic acid as a white solid (544 mg, 25%).

1 H NMR (500 MHz, DMSO-d6) δ 1.41 (s, 6H), 6.48 (s, 1H), 6.88 (t, J = 7.3 Hz,

1H), 7.13 – 7.28 (m, 2H), 7.35 (d, J = 7.4 Hz, 2H), 8.50 (s, 1H).

13C NMR (126 MHz, DMSO) δ 25.38, 54.75, 117.42, 121.02, 128.63, 140.31,

154.29, 176.26.

mp 185–186 oC; (lit. mp 157–158 oC)15(lit. mp 165–166oC)16(lit. mp 117–118 oC)16

Anal Calcd for C11H14N2O3: C, 59.45; H, 6.35; N, 12.60; Found: C, 59.48; H, 6.42;

N, 12.80.

CAS Registry Number 392715-74-1, reported mps are different, commercially available

Synthesis of UC03.

To a solution of 4-fluoro-3-(trifluoromethyl)aniline (4.83 g, 27 mmol) in EtOAc (50 mL) at 0 °C, was added a solution of triphosgene (2.81 g, 9.5 mmol) in toluene (100 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- 108

fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

To a solution of 2-aminoacetic acid (2.25 g, 30 mmol) in 2 M NaOH (50 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (5.53 g, 27 mmol) in CH3CN (5 mL). The mixture was stirred at rt overnight. The insoluble diaryl urea side product in the reaction mixture was removed by filtration. The filtrate was adjusted to pH=3.0 with 2 M

HCl. The resulting precipitate was collected by filtration. The crude material was purified by silica gel chromatography with successive elution with hexane (100 mL),

EtOAc/hexane (60 mL / 60 mL), EtOAc (120 mL) and ethanol (200 mL). The crude product was further purified by crystallization from a mixture of EtOAc (1 mL) and hexane (10 mL) to afford 2-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]acetic acid as a white solid (589 mg, 8%).

1 H NMR (500 MHz, DMSO-d6) δ 3.79 (d, J = 5.8 Hz, 2H), 6.50 (t, J = 5.8 Hz, 1H),

7.38 (t, J = 9.8 Hz, 1H), 7.48 – 7.67 (m, 1H), 7.96 (dd, J = 6.5, 2.8 Hz, 1H), 9.16 (s, 1H),

12.58 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 41.33, 115.15 (q, J = 5.0 Hz), 116.24 (qd, J =

32.3, 13.0 Hz), 117.44 (d, J = 21.3 Hz), 122.64 (q, J = 272.2 Hz), 123.34 (d, J = 8.0 Hz),

137.19, 153.16 (d, J = 246.9 Hz), 155.06, 171.95.

mp 151–153 oC

Anal Calcd for C10H8F4N2O3: C, 42.87; H, 2.88; N, 10.00; Found: C, 43.05; H,

3.00; N, 10.21.

CAS Registry Number 1282316-91-9, no mp data, commercial available

Synthesis of UC04. 109

To a solution of 3-[4-fluoro-3-(trifluoromethyl)phenyl]-5,5-dimethylimidazolidine-

2,4-dione (2.1 g, 7.2mmol) in DMF (30 mL) was added 60% NaH (900 mg, 22mmol) in

DMF (15 mL) at 0 oC under argon. The mixture was then stirred at rt for 2 h and treated dropwise with 1-bromo-3-chloropropane (1695 mg, 10.8 mmol). The mixture was stirred at rt for 2 days. The reaction was adjusted to pH=3.0 with acetic acid. After addition of

H2O (500 mL), the resulting precipitate was collected by filtration and then crystallized from CH2Cl2 (2 mL) to afford 2-[3-[4-fluoro-3-(trifluoromethyl)phenyl]-2- oxotetrahydropyrimidin-1(2H)-yl]-2-methylpropanoic acid as a white solid (1.059 g, 42%).

mp 234–235 oC

1 H NMR (500 MHz, DMSO-d6) δ 1.36 (s, 6H), 2.04 (p, J = 5.9 Hz, 2H), 3.41 (t, J =

6.0 Hz, 2H), 3.64 (t, J = 5.7 Hz, 2H), 7.45 (t, J = 9.7 Hz, 1H), 7.52 – 7.61 (m, 1H), 7.64

(dd, J = 6.6, 2.7 Hz, 1H), 11.83 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 22.46, 23.40, 41.11, 47.38, 60.25, 115.91 (qd, J

= 32.3, 13.1 Hz), 116.90 (d, J = 21.2 Hz), 122.49 (q, J = 272.0 Hz), 123.68 (q, J = 4.2

Hz), 130.99 (d, J = 8.7 Hz), 140.75 (d, J = 3.2 Hz), 153.92, 155.18 (d, J = 250.0 Hz),

175.53.

Anal Calcd for C15H16F4N2O3: C, 51.73; H, 4.63; N, 8.04; Found: C, 52.00; H, 4.87;

N, 8.01.

Synthesis of UC05.

Successful route: 110

The mixture of 4-fluoro-3-(trifluoromethyl)aniline (537 mg, 3 mmol) and 3,3- dimethyldihydrofuran-2,5-dione (512 mg, 4 mmol) in THF (10 mL) was stirred at rt overnight. The solvent was evaporated in vacuo. The residue was then crystallized from

CH2Cl2 (10 mL) to afford 4-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-2,2-dimethyl-4- oxobutanoic acid as a white solid (852 mg, 93%)

1 H NMR (500 MHz, DMSO-d6) δ 1.20 (s, 6H), 2.59 (s, 2H), 7.45 (t, J = 9.8 Hz,

1H), 7.64 – 7.86 (m, 1H), 8.10 (dd, J = 6.6, 2.6 Hz, 1H), 10.25 (s, 1H), 12.10 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 25.26, 45.57, 116.30 (qd, J = 32.3, 13.3 Hz),

116.67 (q, J = 4.8 Hz), 117.62 (d, J = 21.4 Hz), 122.51 (q, J = 272.1 Hz), 124.84 (d, J =

8.1 Hz), 135.91 (d, J = 3.0 Hz), 154.13 (d, J = 248.3 Hz), 169.64, 178.00.

mp 185–186 oC

CAS Registry Number 1492087-76-9, no mp data, commercial available

Failed route:

To a solution of 4-methoxy-3,3-dimethyl-4-oxobutanoic acid (320mg, 2.0 mmol) and DMF (8 µL) in CH2Cl2 (2 mL) was added SOCl2 (0.3 mL) at rt. The mixture was then 111

stirred at rt overnight. The solvent was removed in vacuo to give methyl 4-chloro-2,2- dimethyl-4-oxobutanoate, which was used for the next step without further purification.

To a solution of 4-fluoro-3-(trifluoromethyl)aniline (537 mg, 3 mmol) in DMA (5 mL) was added methyl 4-chloro-2,2-dimethyl-4-oxobutanoate (356 mg, 2.0 mmol) at rt.

The mixture was then stirred at rt overnight. After addition of H2O (100 mL), the resulting precipitate was filtered and purified by chromatography (sg, CH2Cl2 300 mL) followed by crystallization from a mixture of CH2Cl2 (1 mL) and hexane (50 mL) to afford methyl 4-

[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-2,2-dimethyl-4-oxobutanoate as a white solid

(346 mg, 54%).

1 H NMR (500 MHz, DMSO-d6) δ 1.21 (s, 6H), 2.63 (s, 2H), 3.59 (s, 3H), 7.45 (t, J

= 9.8 Hz, 1H), 7.65 – 7.84 (m, 1H), 8.06 (dd, J = 6.6, 2.1 Hz, 1H), 10.29 (s, 1H).

To a solution of methyl 4-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-2,2-dimethyl-

4-oxobutanoate (321mg, 1.0mmol) in MeOH (8 mL) was added a solution of NaOH (160 mg) in H2O (4 mL) at rt. The mixture was then stirred at rt overnight. The reaction was adjusted to pH=3.0 with 32% HCl. The solvent was removed in vacuo. After addition of

H2O (10 mL), the resulting precipitate was filtered and rinsed with H2O (10 mL) to afford a mixture of 4-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-2,2-dimethyl-4-oxobutanoic acid and 4-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-3,3-dimethyl-4-oxobutanoic acid (1:1) as a white solid.

Synthesis of UC06. 112

To a solution of 2-[4-fluoro-3-(trifluoromethyl)phenyl]acetic acid (2.2 g, 10 mmol) and DMF (40 µL) in CH2Cl2 (10 mL) was added SOCl2 (1.5 mL) at rt. The mixture was then stirred at rt overnight. The solvent was removed in vacuo to give 2-[4-fluoro-3-

(trifluoromethyl)phenyl]acetyl chloride, which was used for the next step without further purification.

To a solution of 2-amino-2-methylpropanoic acid (1030 mg, 10 mmol) in 2 M

NaOH (10 mL) was added 2-[4-fluoro-3-(trifluoromethyl)phenyl]acetyl chloride (2.4 g, 10 mmol) in 2 M NaOH (5 mL) at 0 oC dropwise. The mixture was then stirred at 0 oC for 2 h.

The reaction was adjusted to pH=2 with 32% HCl. The resulting precipitate was filtered and crystallized from CH2Cl2 (10 mL) to afford 2-[2-[4-fluoro-3-

(trifluoromethyl)phenyl]acetamido]-2-methylpropanoic acid (1.358 g, 44%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 1.34 (s, 6H), 3.52 (s, 2H), 7.44 (dd, J = 10.8, 8.6

Hz, 1H), 7.51–7.62 (m, 1H), 7.65 (dd, J = 7.2, 2.1 Hz, 1H), 8.37 (s, 1H), 12.18 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 24.86, 40.41, 54.92, 116.08 (qd, J = 32.1, 12.3

Hz), 116.81 (d, J = 20.4 Hz), 122.70 (q, J = 271.8 Hz), 127.42 (q, J = 4.5 Hz), 133.54 (d,

J = 3.8 Hz), 135.65 (d, J = 8.5 Hz), 157.58 (dq, J = 251.8, 2.8 Hz), 168.77, 175.33.

mp 190–191 oC

Synthesis of UC07. 113

To a solution of triphosgene (3.680 g, 12.4 mmol) in toluene (100 mL) at 0 °C was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (6.300 g, 35.2 mmol) in EtOAc

(100 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 70 oC overnight. The solvents were evaporated in vacuo to afford 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

To a solution of 1-aminocyclopropanecarboxylic acid (3.066 g, 30.4 mmol) in 1 M

NaOH (10 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (7.216 g,

o o 35.2 mmol) in CH3CN (10 mL) at 0 C dropwise. The mixture was stirred at 0 C for 3 h and then was warmed to rt overnight. The reaction mixture was concentrated and then redissolved in 1 M NaOH (50 mL). The resulting precipitate, insoluble diaryl urea side product, was removed by filtration. The filtrate was adjusted to pH=3.0 with 32% HCl.

The resulting precipitate was collected by filtration to give 1-[3-[4-fluoro-3-

(trifluoromethyl)phenyl]ureido]cyclopropanecarboxylic acid as a white solid (4274 mg,

46%).

1 H NMR (500 MHz, DMSO-d6) δ 1.04 (dd, J = 7.7, 4.5 Hz, 2H), 1.35 (dd, J =7.7,

4.5 Hz, 2H), 7.18 (s, 1H), 7.37 (t, J = 9.8 Hz, 1H), 7.48 – 7.66 (m, 1H), 8.00 (dd, J = 6.5,

2.7 Hz, 1H), 9.49 (s, 1H), 12.31 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 16.84, 32.97, 114.99, 116.17 (qd, J = 31.9, 12.9

Hz), 117.37 (d, J = 21.3 Hz), 122.67 (q, J = 272.0 Hz), 123.22 (d, J = 5.9 Hz), 137.33 (d,

J = 2.7 Hz), 153.06 (d, J = 247.8 Hz), 155.47, 174.46. 114

mp: 201-202 oC

Anal Calcd for C12H10F4N2O3·0.7H2O: C, 45.21; H, 3.60; N, 8.79; Found: C, 45.06;

H, 3.29; N, 8.72.

Synthesis of UC08.

To a solution of triphosgene (4.144 g, 14 mmol) in toluene (150 mL) at 0 °C was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (7.16 g, 40 mmol) in EtOAc (120 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 70 oC overnight. The solvents were evaporated in vacuo to afford 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

To a solution of 1-aminocyclobutanecarboxylic acid (1.035 g, 9 mmol) in 1 M

NaOH (15 mL) was added 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (2.05 g, 10

o o mmol) in CH3CN (10 mL) at 0 C dropwise. The mixture was stirred at 0 C for 3 h and then was warmed to rt overnight. The reaction mixture was concentrated and the resulting precipitate, insoluble diaryl urea side product, was removed by filtration. The filtrate was adjusted to pH=3.0 with 32% HCl. The resulting precipitate was collected by filtration and then recrystallized from diethyl ether (5 mL) to give 1-[3-[4-fluoro-3-

(trifluoromethyl)phenyl]ureido]cyclobutanecarboxylic acid as a white solid (1.177 g, 41%). 115

1 H NMR (500 MHz, DMSO-d6) δ 1.85–2.02 (m, 2H), 2.16 – 2.29 (m, 2H), 2.43–

2.60 (m, 2H), 7.00 (s, 1H), 7.37 (t, J = 9.7 Hz, 1H), 7.44–7.64 (m, 1H), 7.99 (dd, J = 6.6,

2.7 Hz, 1H), 8.98 (s, 1H), 12.45 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 15.00, 31.00, 57.38, 115.11 (q, J = 5.0 Hz),

116.28 (qd, J = 32.0, 12.8 Hz), 117.42 (d, J = 21.3 Hz), 122.69 (q, J = 271.9 Hz), 123.36

(d, J = 7.8 Hz), 137.15 (d, J = 2.6 Hz), 153.19 (dq, J = 247.1, 2.3 Hz), 154.34, 175.20.

mp: 175-176 oC

Anal Calcd for C13H12F4N2O3: C, 48.76; H, 3.78; N, 8.75; Found: C, 48.51; H, 3.94;

N, 8.88.

116

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Karlberg, T.; Spjut, S.; Linusson, A.; Schüler, H.; Elofsson, M. Towards small molecule inhibitors of mono-ADP-ribosyltransferases. Eur. J. Med. Chem. 2015, 95, 546-551.

14. Buijs, W.; Comba, P.; Corneli, D.; Pritzkow, H. Structural and mechanistic studies of the copper (II)-assisted ortho-hydroxylation of benzoates by trimethylamine N-oxide. J.

Organomet. Chem. 2002, 641, 71-80.

15. Durini, M.; Russotto, E.; Pignataro, L.; Reiser, O.; Piarulli, U. SupraBox: Chiral

Supramolecular Oxazoline Ligands. Eur. J. Org. Chem. 2012, 2012, 5451-5461.

16. Ivanov, P.; Pojarlieff, I.; Blagoeva, I.; Jaime, C.; Angelova, V.; Koedjikov, A. A large gem‐dimethyl effect in the cyclization of ω‐phenylhydantoic acids: computational modeling of the gem ‐dimethyl effect on the acid ‐ or base‐ catalyzed cyclization of hydantoic acids and esters. J. Phys. Org. Chem. 2004, 17, 423-430.

118

Chapter 5.

N,N’-diaryl urea antischistosomal agents

Abstract

Novel N,N'-diaryl ureas were designed and synthesized as new analogues of Ro

13-3978, some of which showed significant antischistosomal effects. The most promising compound identified in this work was N,N’-di(quinoxalin-2-yl)urea. This new lead compound can serve as a new direction for further optimization. In addition, various synthetic routes to diaryl ureas were described.

5.1. Introduction

Schistosomiasis is an intravascular disease caused by parasitic trematode worms of the genus Schistosoma. The burden of disease attributable to the three major human schistosome species (Schistosoma mansoni, S. Haematobium, and S. japonicum) is estimated to be almost 200 million infected patients1. More than 90% of the roughly

200 million cases of schistosomiasis occur in Africa2, 3 and close to 800 million are at risk.

It is one of the most prevalent of the tropical infectious diseases4. In the absence of vaccines5 and with the challenges surrounding implementation of other preventive measures such as access to safe water and improved sanitation, the current and precarious mainstay for treatment and control of schistosomiasis is praziquantel (PZQ)6,

7. This dependence on a single drug is alarming, as there is some evidence that resistance to PZQ may be emerging in Africa and laboratory studies indicate that schistosomes exposed to repeated in vivo PZQ treatments have reduced drug sensitivities8, 9. Furthermore, PZQ is inactive against the juvenile schistosomula; this may be an important factor in the frequently observed treatment ‘failures’ in areas endemic for schistosomiasis10. Even so, drug discovery for schistosomiasis has 119

languished11-13, although several antischistosomal lead compounds14, 15 have been identified in recent years.

Ingram-Sieber recently screened the MMV malaria box of 400 commercially available malaria active compounds for antischistosomal activity16. This library was assayed initially against schistosomula and then selected compounds progressed to ex vivo and in vivo evaluation against adult schistosomes. The most promising lead identified in this work was N,N’-diarylurea MMV665852. It was nearly as potent as

PZQagainst ex vivo S. mansoni. A single 400 mg/kg oral dose of MMV665852 administered to S. mansoni-infected mice reduced adult WBR by 53%.16 Previously, we had tested the symmetrical N,N’-diarylurea AR33 in S.mansoni-infected mice, as AR33 was a side product formed in the synthesis of the aryl hydantoins. A single 100 mg/kg oral dose of AR33 administered to S. mansoni-infected mice reduced adult WBR by 40% indicating that this N,N’-diarylurea is more effective than the lead N,N’-diarylurea

MMV665852. Thus, this very simple compound offers intriguing possibilities for further optimization.

5.2. Design

With the goal of increasing the antischistosomal activity and the solubility of the hydrophobic N,N’-diarylurea lead compounds, an initial library of N,N’-diarylureas and closely related compounds that could accelerate the SAR for this chemotype and help 120

focus future medicinal chemistry efforts was designed. They include the symmetrical diarylureas, unsymmetrical diarylureas and phenylbenzamides.

Most target compounds maintain the 4-fluoro-3-(trifluoromethyl)phenyl core structure of AR33 while involving carboxyl U13 and U16, pyridyl U05, U10 and U11, quinoxalinyl U09, isoxazolyl U17, amide U15 and U18, and cyano U14 to increase solubility and polarity. AR42 and ARX contain substructure of praziquantel. AR45, AR46 and AR47 are analogs of anthelmintic niclosamide. U03 and U04 keep the core structure of active compound AR43. Symmetrical ureas AR50, U07 and U08 are potential control compounds. The final set of target compounds maintain the 4-fluoro-3-

(trifluoromethyl)phenyl substructure with various central urea substructures: conformationally-restricted AR41 and size-extension AR39.

5.3. Chemistry

Target compounds AR33-U18 were obtained in variable yields by reaction between aryl isocyanates ISO-1 or ISO-2 and anilines AR33-1-U18-1 or praziquanamine

(AR42-1) in different solvents to form the corresponding unsymmetrical urea (scheme

5.1. and scheme 5.4.)

Exposure of U07-1 or U08-1 to CDI at rt effected carbonylation to give corresponding symmetric ureas U07 or U08 (scheme 5.2.).

N-arylation via palladium promoted cross-coupling of aryl iodide AR41-1 with 1,3- ethyleneurea (AR41-2) afforded conformationally restricted diaryl urea AR41. Symmetric diaryl ureas U04 and AR50 were obtained in a two-step sequence by reactions between pyridylamine U04-1 or aminoquinoxaline AR50-1 and triphosgene to form the corresponding isocyanates, which were further reacted with U04-1 or AR50-1 to provide

U04 and AR50 respectively (Scheme 5.3.). 121

Salicylanilides AR45-AR47 were obtained by the acylation of corresponding anilines AR45-1, AR46-1 or AR47-1 with 5-chloro-2-hydroxybenzoyl chloride (BC-1) respectively (scheme 5.5.).

Target compound U03 was synthesized via EDCI-mediated coupling of pyridyl carboxylic acid U03-1 with pyridylamine U04-1 in DMA (scheme 5.6.).

AR39 was obtained in one step by reaction between terephthaloyl chloride

(AR39-2) and aniline AR39-1 under basic condition (scheme 5.7.).

122

Scheme 5.1.

o (a) CH2Cl2, CH3CN, or THF, rt to 80 C, 4-22 h.

123

Scheme 5.2.

(a) CDI, THF, rt, 48 h.

Scheme 5.3.

(a) Cs2CO3, toluene, Xantphos, pd2(dba)3, DMA, rt, 24 h;

o (b) Triphosgene, DIPEA,CH2Cl2, 0 C to rt, 12 h;

(d) Triphosgene, toluene, EtOAc, 0 oC-85 oC, 24 h; 124

(e) 2 M NaOH.

Scheme 5.4.

(a) CH2Cl2, rt, 12-20 h.

125

Scheme 5.5.

(a) CH2Cl2, DMAP, Et3N, rt, 12 h.

126

Scheme 5.6.

(a) EDCI•HCl, DMA, rt, 12 h.

Scheme 5.7.

(a) Et3N, DMA, rt, 24 h.

127

5.4. Results and Discussion

This library of urea analogs was assayed initially against NTS and adult worms

(in vitro) then selected compounds progressed to in vivo evaluation against adult schistosomes. The most promising compound identified in this work was AR50. As shown in Table 5.1., a single 200 mg/kg oral dose of AR50 administered to S. mansoni- infected mice reduced adult WBR by 75.1%, while a dose of 100 mg/kg reduced WBR by 62%. Thus, this very simple compound offers intriguing possibilities for further optimization, although this is tempered by its large PSA of 92.7 Å2.

Unsymmetrical diaryl ureas, AR38, AR49, U05 and U14, maintain the 4-fluoro-3-

(trifluoromethyl)aniline core of AR33 and involve dichlorophenyl (AR38), cyanophenyl

(U14), quinoxalinyl (AR49) or pyridyl (U05). These compounds demonstrated in vivo activity at 400 mg/kg.

Salicylanilide compounds were toxic. Two mice, treated with AR46, died 1 and 4 hours after application. Although the salicylanilides AR45, AR46, and AR47 were active in vitro, they were not active in vivo. We suggest that the closely related drug niclosamide is not absorbed from the small intestine. Thus, salicylanilides are not a good compound series.

5.5. Summary

In conclusion, we designed an initial library of N,N’-diarylureas and closely related compounds that could accelerate the SAR for this chemotype and help focus future med-chem efforts. The most promising compound identified in this work was N,N’- di(quinoxalin-2-yl)urea (AR50).

128

Table 5.1. In vitro, in vivo antischistosomal data for urea analogs

in vitro in vitro in vivo in vivo IC50 IC50 WBR WBR COMPOUND STRUCTURE against against % % NTS adult 400 100 (µM) (µM) mg/kg mg/kg

AR33 0.15 0.19 30.7 40

AR38 0.13 0.23 53.4 0

AR39 >33 >10 54

AR41 >33 >10 0

AR42 0

ARX

AR45 0.08 0.001 0

AR46 0.01 0.33 toxic

129

AR47 0.09 0.007 27.5

AR49 >33 >10 44.9

AR50 >33 >10 75.1a 62

U03 >33 >10

U04 2.43 0.88 0

U05 >33 >10 46.1

U07 >33

U08 >33

U09 >33

U10 >10 >33

U11 5.595 33

U13 >33

130

U14 >10 0.94 43.5

U15 >10 >10

U16 >10 >10

U17 >10 >10

U18 >33 >10

a. 200 mg/kg po.

131

5.6 Experimental

5.6.1. General.

Melting points are uncorrected. 1D 1H and 13C NMR spectra were recorded on a 500

MHz spectrometer using CDCl3or DMSO-d6 as solvents. All chemical shifts are reported

1 in parts per million (ppm) and are relative to internal (CH3)4Si (0 ppm) for H and CDCl3

13 (77.2 ppm) or DMSO-d6 (39.5 ppm)for C NMR. EI GC-MS data were obtained using a quadrapole mass spectrometer with 30 m DB-XLB type columns and a He flow rate of 1 mL/min. Silica gel (sg) particle size 32−63 μm was used for all flash column chromatography. Reported reaction temperatures are those of the oil bath.

5.6.2. Synthesis

1. Synthesis of AR33.

A mixture of 4-fluoro-3-(trifluoromethyl)aniline (1.79 g, 10 mmol) and 1-fluoro-4- isocyanato-2-(trifluoromethyl)benzene (2.05 g, 10 mmol) in CH3CN (10 mL) was stirred at rt overnight. Evaporation gave a crude product which was further purified by successive recrystallizations from H2O (20 mL) and a mixture of EtOAc (3 mL) and hexane (5 mL) to afford 1,3-bis[4-fluoro-3-(trifluoromethyl)phenyl]urea (1.501 g, 39%) as a white solid. mp: 198-199 oC 132

1 H NMR (500 MHz, DMSO-d6) δ 7.45 (t, J = 9.8 Hz, 2H), 7.60–7.75 (m, 2H), 7.98 (dd, J

= 6.7, 2.6 Hz, 2H), 9.17 (s, 2H).

13 C NMR (126 MHz, DMSO-d6) δ 116.38 (q, J = 5.0 Hz), 116.67 (qd, J = 32.1, 12.9 Hz),

117.33 (d, J = 21.7 Hz), 122.70 (q, J = 271.9 Hz), 124.36 (d, J = 8.1 Hz), 136.34 (d, J =

2.7 Hz), 152.69, 153.97 (d, J = 248.7 Hz).

Anal Calcd for C15H8F8N2O: C, 46.89; H, 2.10; N, 7.29; Found: C, 47.00; H, 2.37; N, 7.34

CAS Registry Number 35685-20-2, No mp data.

2. Synthesis of AR38.

To a solution of 1,2-dichloro-4-isocyanatobenzene (1.710 g, 9.1 mmol) in CH2Cl2 (100 mL) was added 4-fluoro-3-(trifluoromethyl)aniline (2.443 g, 13.6 mmol). The reaction mixture was stirred at rt for 20 h. The precipitation was collected by filtration and washed with CH2Cl2 (10 mL) to afford 1-(3,4-dichlorophenyl)-3-[4-fluoro-3-

(trifluoromethyl)phenyl]urea as (3.000 g, 90%) a white solid. mp 234–235 oC

1 H NMR (500 MHz, DMSO-d6) δ 7.37 (dd, J = 8.8, 2.5 Hz, 1H), 7.44 (t, J = 9.8 Hz, 1H),

7.52 (d, J = 8.8 Hz, 1H), 7.60–7.71 (m, 1H), 7.88 (d, J = 2.4 Hz, 1H), 7.99 (dd, J = 6.6,

2.6 Hz, 1H), 9.12 (s, 1H), 9.16 (s, 1H). 133

13 C NMR (125 MHz, DMSO-d6) δ 116.36 (d, J = 5.2 Hz), 116.39 (qd, J = 32.2, 13.1 Hz),

117.57 (d, J = 21.4 Hz), 118.64, 119.63, 122.57 (q, J = 271.9 Hz), 123.49, 124.59 (d, J =

8.0 Hz), 130.53, 131.03, 136.17 (d, J = 2.7 Hz), 139.63, 152.38, 153.82 (d, J = 248.0 Hz).

Anal Calcd for C14H8Cl2F4N2O: C, 45.80; H, 2.20; N, 7.63; Found: C, 46.12; H, 2.21; N,

7.89.

CAS Registry Number 799-22-4, no mp data, commercial available

3. Synthesis of AR39.

To a solution of 4-fluoro-3-(trifluoromethyl)aniline (3.943 g, 22 mmol) and TEA (4.100 g,

40 mmol) in DMA (40 mL) was added terephthaloyl dichloride (2.020 g, 10 mmol) at rt.

After it was stirred at rt for 24 h, the reaction mixture was poured into water (200 mL).

The precipitation was collected by filtration and washed with CHCl3 (10 mL) to afford

N1,N4-bis[4-fluoro-3-(trifluoromethyl)phenyl]terephthalamide (4.293 g, 88%) as a white solid. mp 305–306 oC

1 H NMR (500 MHz, DMSO-d6) δ 7.56 (t, J = 9.8 Hz, 2H), 8.06–8.18 (m, 6H), 8.28 (dd, J

= 6.6, 2.8 Hz, 2H), 10.73 (s, 2H).

13 C NMR (125 MHz, DMSO-d6) δ 116.45 (qd, J = 32.4, 13.1 Hz), 117.51 (d, J = 21.5 Hz),

118.52 (d, J = 5.2 Hz), 122.56 (q, J = 271.8 Hz), 126.43 (d, J = 8.3 Hz), 127.80, 135.77

(d, J = 3.0 Hz), 137.11, 154.78 (d, J = 250.5 Hz), 164.99. 134

Anal Calcd for C22H12F8N2O2·1.5 H2O: C, 51.27; H, 2.93; N, 5.44; Found: C, 51.66; H,

3.21; N, 5.00.

4. Synthesis of AR41.

To a solution of 1-fluoro-4-iodo-2-(trifluoromethyl)benzene (1500 mg, 5.2 mmol), imidazolidin-2-one (344 mg, 4 mmol) and Cs2CO3 (2.700 g, 8.3 mmol) in toluene (46 mL) were added xantphos (120 mg, 0.2 mmol) and pd2(dba)3 (93 mg, 0.1 mmol). After it was heated at 80oC under argon overnight, the solvent was evaporated in vacuo. The residue was purified by chromatography (EtOAc:hexane 1:1) to afford 1,3-bis[4-fluoro-3-

(trifluoromethyl)phenyl]imidazolidin-2-one (345 mg, 21%) as a white solid. mp 153–154 oC

1 H NMR (500 MHz, DMSO-d6) δ 4.05 (s, 4H), 7.56 (t, J = 9.8 Hz, 2H), 7.77–7.91 (m, 2H),

8.14 (dd, J = 6.5, 2.8 Hz, 2H).

13 C NMR (126 MHz, DMSO-d6) δ 41.45, 115.92 (q, J = 4.9 Hz), 116.45 (qd, J = 32.2,

13.1 Hz), 117.61 (d, J = 21.4 Hz), 122.70 (q, J = 272.1 Hz), 123.85 (d, J = 8.0 Hz),

136.56 (d, J = 2.7 Hz), 154.11 (dq, J = 249.3, 2.2 Hz), 154.34.

GC-MS purity: 100%

Anal Calcd for C17H10F8N2O: C, 49.77; H, 2.46; N, 6.83; Found: C, 50.00; H, 2.71; N,

6.55. 135

5. Synthesis of AR42.

The mixture of 1,2,3,6,7,11b-hexahydro-4H-Pyrazino[2,1-a]isoquinolin-4-one(737 mg,

3.64 mmol) and 1-fluoro-4-isocyanato-2-(trifluoromethyl)benzene (498 mg, 2.43 mmol) in

CH2Cl2 (24 mL) was stirred at rt overnight. The precipitation was collected by filtration and washed with CH2Cl2 (1 mL) to afford N-[4-fluoro-3-(trifluoromethyl)phenyl]-4-oxo-

3,4,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinoline-2(11bH)-carboxamide (459 mg, 46%) as a white solid. mp 227–228 oC

1 H NMR (500 MHz, DMSO-d6) δ 2.74–2.95 (m, 3H), 3.14 (dd, J = 13.2, 10.6 Hz, 1H),

3.96 (d, J = 17.3 Hz, 1H), 4.44 (d, J = 17.3 Hz, 1H), 4.57 (dt, J = 11.4, 3.6 Hz, 1H), 4.62–

4.72 (m, 1H), 4.97 (dd, J = 10.6, 4.2 Hz, 1H), 7.20–7.34 (m, 3H), 7.38–7.47 (m, 2H), 7.83

(dt, J = 7.9, 3.7 Hz, 1H), 7.97 (dd, J = 6.5, 2.7 Hz, 1H), 9.05 (s, 1H).

13 C NMR (126 MHz, DMSO-d6) δ 28.23, 38.24, 46.91, 47.54, 54.23, 116.09 (qd, J = 32.0,

12.8 Hz), 117.25 (d, J = 21.1 Hz), 117.55 (d, J = 5.4 Hz), 122.69 (q, J = 272.3 Hz),

125.53 (d, J = 7.9 Hz), 125.65, 126.58, 127.09, 129.08, 133.40, 135.10, 136.98 (d, J =

2.8 Hz), 153.82 (d, J = 248.3 Hz), 154.03, 164.52.

Anal Calcd for C20H17F4N3O2: C, 58.97; H, 4.21; N, 10.32; Found: C, 59.19; H, 4.47; N,

10.53. 136

6. Synthesis of AR45.

To a solution of 5-chloro-2-hydroxybenzoic acid (1.7 g, 9.9 mmol) in toluene (5 mL) was added thionyl chloride (5 mL).The reaction mixture was heated to 70 oC overnight.

The solvent was then evaporated in vacuo to afford 5-chloro-2-hydroxybenzoyl chloride which was used for the next step without further purification.

To a solution of 5-chloro-2-hydroxybenzoyl chloride in CH2Cl2 (10 mL) was added a mixture of 4-fluoro-3-(trifluoromethyl)aniline (1.180 g, 6.6 mmol), DMAP (85.4 mg, 0.7 mmol) and Et3N (1.3 g, 13 mmol). After stirring at rt overnight, the solvent was evaporated in vacuo. The residue was purified by successive crystallization from a mixture of CH3CN (3 mL) and H2O (30 mL) and a mixture of DMSO (2 mL) and H2O (20 mL) to afford 5-chloro-N-[4-fluoro-3-(trifluoromethyl)phenyl]-2-hydroxybenzamide (0.569 g, 24%) as a white solid. mp 197–198 oC (lit. mp 203–205 oC)17

1 H NMR (500 MHz, DMSO-d6) δ 7.03 (d, J = 8.8 Hz, 1H), 7.48 (dd, J = 8.9, 2.7 Hz, 1H),

7.55 (t, J = 9.8 Hz, 1H), 7.88 (d, J = 2.7 Hz, 1H), 7.96–8.13 (m, 1H), 8.22 (dd, J = 6.7,

2.6 Hz, 1H), 10.65 (s, 1H), 11.56 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 116.43 (qd, J = 32.1, 13.0 Hz), 117.65 (d, J = 21.4 Hz),

118.75 (d, J = 5.0 Hz), 119.08, 119.78, 122.48 (q, J = 271.9 Hz), 122.62, 126.82 (d, J =

8.3 Hz), 128.39, 133.10, 134.97 (d, J = 3.0 Hz), 154.92 (d, J = 250.6 Hz), 156.68, 165.21. 137

Anal Calcd for C14H8ClF4NO2: C, 50.40; H, 2.42; N, 4.20; Found: C, 50.50; H, 2.68; N,

4.30.

7. Synthesis of AR46.

A solution of 5-chloro-2-hydroxybenzoic acid (1.7 g, 10 mmol) and SOCl2 (5 mL) in toluene (5 mL) was stirred at 70 oC overnight. The mixture was concentrated to afford the 5-chloro-2-hydroxybenzoyl chloride which was used for the next step without further purification. To a solution of 3,5-bis(trifluoromethyl)aniline (1.526 g, 6.6 mmol) in CH2Cl2

(10 mL) were added the acid chloride, Et3N (1.3 g, 13 mmol), and DMAP (85.4 mg, 0.7 mmol). The mixture was stirred at room temperature overnight. After removal of CH2Cl2 under reduced pressure, the residue was recrystallized from a mixture of CH3CN (4 mL) and H2O (30 mL) to afford N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2- hydroxybenzamide (0.73 g, 29%) as a white solid. mp 175–177 oC; (lit. mp 172–173 oC)17

1 H NMR (500 MHz, DMSO-d6) δ 7.04 (d, J = 8.8 Hz, 1H), 7.49 (dd, J = 8.8, 2.7 Hz, 1H),

7.85 (s, 1H), 7.86 (d, J = 2.7 Hz, 1H), 8.45 (s, 2H), 10.94 (s, 1H), 11.40 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 116.79, 119.15, 120.15, 120.26, 122.45, 123.21 (q, J =

272.9 Hz), 128.55, 130.70 (q, J = 32.9 Hz), 133.18, 140.31, 156.66, 165.51. 138

Anal Calcd for C15H8ClF6NO2: C, 46.96; H, 2.10; N, 3.65; Found: C, 47.12; H, 2.21; N,

3.85.

8. Synthesis of AR47.

To a solution of 5-chloro-2-hydroxybenzoic acid (3.5 g, 20.3 mmol) in toluene (10 mL) was added thionyl chloride (10 mL).The reaction mixture was heated to 70 oC overnight.

The solvent was then evaporated in vacuo to afford 5-chloro-2-hydroxybenzoyl chloride which was used for the next step without further purification.

To a solution of 5-chloro-2-hydroxybenzoyl chloride in CH2Cl2 (50 mL) was added a mixture of 3,4-dichloroaniline (2.1 g, 12.9 mmol), DMAP (175 mg, 1.5 mmol) and Et3N

(2.6 g, 26 mmol). After stirring at rt overnight, the solvent was evaporated in vacuo. The residue was purified by crystallization from a mixture of acetic acid (1 mL), CH3CN (1 mL) and H2O (10 mL), and then successive recrystallization from a mixture of xylene (1 mL) and hexane (10 mL) and a mixture of DMSO (2 mL) and H2O (20 mL) to afford 5-chloro-

N-(3,4-dichlorophenyl)-2-hydroxybenzamide (1.574 g, 39%) as a white solid. mp 243–245 oC; (lit. mp 246–248 oC)18

1 H NMR (500 MHz, DMSO-d6) δ 7.03 (d, J = 8.8 Hz, 1H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H),

7.62 (d, J = 8.8 Hz, 1H), 7.68 (dd, J = 8.8, 2.4 Hz, 1H), 7.87 (d, J = 2.7 Hz, 1H), 8.11 (d,

J = 2.3 Hz, 1H), 10.56 (s, 1H), 11.56 (s, 1H). 139

13 C NMR (125 MHz, DMSO-d6) δ 119.00, 120.09, 120.57, 121.76, 122.73, 125.65,

128.49, 130.63, 130.97, 133.05, 138.31, 156.32, 164.98.

Anal Calcd for C13H8Cl3NO2: C, 49.32; H, 2.55; N, 4.42; Found: C, 49.41; H, 2.68; N,

4.53

9. Synthesis of AR49.

To a solution of triphosgene (1 g, 3.4 mmol) in toluene (40 mL) at 0 oC was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (1.79 g, 10 mmol) in CH2Cl2 (20 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

A mixture of quinoxalin-2-amine (0.5 g, 3.4 mmol) and 1-fluoro-4-isocyanato-2-

(trifluoromethyl)benzene (2.05 g, 10 mmol) in CH3CN (20 mL) was refluxed overnight.

After cooling to rt, the resulting precipitate was filtered and crystallized from hot methanol to afford 1-[4-fluoro-3-(trifluoromethyl)phenyl]-3-(quinoxalin-2-yl)urea (0.743 g,

61%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.52 (t, J = 9.7 Hz, 1H), 7.68 (ddd, J = 8.3, 7.0, 1.4 Hz,

1H), 7.81 (ddd, J = 8.3, 7.0, 1.5 Hz, 1H), 7.88 (dt, J = 8.0, 3.5 Hz, 1H), 8.03 (ddd, J =

15.5, 8.4, 1.3 Hz, 2H), 8.19 (dd, J = 6.5, 2.8 Hz, 1H), 9.02 (s, 1H), 10.48 (s, 1H), 11.11 (s,

1H). 140

13 C NMR (125 MHz, DMSO-d6) δ 116.52 (qd, J = 32.2, 13.0 Hz), 117.18 (q, J = 5.5 Hz),

117.72 (d, J = 21.5 Hz), 122.53 (q, J = 272.3 Hz), 125.68 (d, J = 8.2 Hz), 126.92, 127.30,

128.63, 130.67, 135.28, 138.21, 138.67, 139.50, 147.24, 152.02, 154.39 (d, J = 249.3

Hz). mp: 270-272 oC

Anal Calcd for C16H10F4N4O: C, 54.86; H, 2.88; N, 16.00; Found: C, 55.00; H, 3.00; N,

16.22

10. Synthesis of AR50.

To a solution of triphosgene (360 mg, 1.22 mmol) in toluene (30 mL) at 0 oC was added a solution of quinoxalin-2-amine (530 mg, 3.65 mmol) in EtOAc (30 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC for 24 h. The solvents were evaporated in vacuo to afford 2- isocyanatoquinoxaline which was used for the next step without further purification.

To a solution of 2-amino-2-methylpropanoic acid (134 mg, 1.3 mmol) in 2 M NaOH (5 mL) was added 2-isocyanatoquinoxaline (625 mg, 3.65 mmol). The reaction mixture was stirred at rt overnight. The precipitated byproduct was filtered to afford 1,3-di(quinoxalin-

2-yl)urea (307 mg, 53%) as a white solid. mp: 303-305 oC 141

1 H NMR (500 MHz, DMSO-d6) δ 7.73 (ddd, J = 8.3, 6.9, 1.4 Hz, 2H), 7.85 (ddd, J = 8.4,

6.9, 1.4 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 8.06 (d, J = 8.3 Hz, 2H), 9.41 (s, 2H), 11.26 (s,

2H).

13C NMR (125 MHz, DMSO) δ 126.95, 127.81, 128.78, 130.89, 138.90, 139.43, 147.03,

151.68.

Anal Calcd for C17H12N6O: C, 64.55; H, 3.82; N, 26.57; Found: C, 64.81; H, 4.00; N,

26.56.

11. Synthesis of U03.

To a solution of 2-(trifluoromethyl)pyridin-4-amine(162 mg, 1 mmol) and 2-

(trifluoromethyl)isonicotinic acid (172 mg, 0.9 mmol) in DMA (4 mL) was added1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (200 mg, 1 mmol) at rt. The mixture was then stirred at rt overnight. After addition of H2O (75 mL), the resulting precipitate was filtered to afford 2-(trifluoromethyl)-N-[2-(trifluoromethyl)pyridin-4- yl]isonicotinamide (138 mg, 46%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 8.04 (dd, J = 5.8, 1.9 Hz, 1H), 8.22 (dd, J = 4.9, 1.6 Hz,

1H), 8.27 (d, J = 2.0 Hz, 1H), 8.39 (d, J = 1.1 Hz, 1H), 8.73 (d, J = 5.5 Hz, 1H), 9.04 (d, J

= 5.0 Hz, 1H), 11.31 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 110.98 (q, J = 3.2 Hz), 116.83, 118.73 (q, J = 2.8 Hz),

121.43 (q, J = 274.5), 121.58 (q, J = 274.8), 125.62, 143.09, 146.70–147.78 (m), 147.13,

151.36, 151.37, 164.03. 142

mp 108–110 oC

Anal Calcd for C13H7F6N3O·0.5H2O: C, 45.36; H, 2.34; N, 12.21; Found: C, 45.81; H,

2.60; N, 11.83.

12. Synthesis of U04.

To a mixture of 2-(trifluoromethyl)pyridin-4-amine (314 mg, 1.9 mmol) and N,N-

o diisopropylethylamine (774 mg, 6 mmol) in CH2Cl2 (30 mL) at 0 C was added a solution of triphosgene (592 mg, 2 mmol) in CH2Cl2 (20 mL) dropwise. The reaction mixture was allowed to return to rt overnight. The solvents were evaporated in vacuo to afford 4- isocyanato-2-(trifluoromethyl)pyridine which was used for the next step without further purification.

To a solution of 4-isocyanato-2-(trifluoromethyl)pyridine (357 mg, 1.9 mmol) and N,N- diisopropylethylamine (238 mg, 1.8 mmol) in CH2Cl2 (10 mL) was added 2-

(trifluoromethyl)pyridin-4-amine (300 mg, 1.8 mmol) at rt. After the mixture was stirred at rt overnight, and additional portion of triphosgene (416mg, 1.4 mmol) and N,N- diisopropylethylamine (548 mg, 4.2 mmol) was added. The mixture was then stirred at rt overnight. The solvent was evaporated in vacuo. The residue was then crystallized from ethanol:H2O (1:3) to afford 1,3-bis[2-(trifluoromethyl)pyridin-4-yl]urea (236 mg, 37%) as a white to pale yellow solid. 143

1 H NMR (500 MHz, DMSO-d6) δ 7.66 (d, J = 4.7 Hz, 2H), 8.07 (s, 2H), 8.59 (d, J = 5.6

Hz, 2H), 9.92 (s, 2H).

13 C NMR (125 MHz, DMSO-d6) δ 109.32, 115.39, 121.61 (q, J = 274.2 Hz), 147.33 (q, J

= 33.2 Hz), 147.64, 150.91, 151.82. mp 205–207 oC

Anal Calcd for C13H8F6N4O·0.3H2O: C, 43.91; H, 2.44; N, 15.75; Found: C, 44.19; H,

2.71; N, 15.33.

13. Synthesis of U05.

To a solution of triphosgene (658 mg, 2.2 mmol) in toluene (8 mL) at 0 oC was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (1.199 g, 6.7 mmol) in EtOAc (6 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

To a solution of pyridin-2-amine (622 mg, 6.6 mmol) in THF (19 mL) was added 1-fluoro-

4-isocyanato-2-(trifluoromethyl)benzene (1.37 g, 6.7 mmol) in THF (7 mL) at rt. The reaction mixture was stirred at 80 °C for 4 h. The solvent was evaporated in vacuo. The residue was then crystallized from EtOAc to afford1-[4-fluoro-3-(trifluoromethyl)phenyl]-

3-(pyridin-2-yl)urea (430 mg, 22%) as a white solid. 144

1 H NMR (500 MHz, DMSO-d6) δ 6.94–7.11 (m, 1H), 7.39–7.54 (m, 2H), 7.69–7.82 (m,

2H), 8.08 (dd, J = 6.6, 2.7 Hz, 1H), 8.30 (d, J = 5.2 Hz, 1H), 9.57 (s, 1H), 10.87 (s, 1H).

13 C NMR (126 MHz, DMSO-d6) δ 112.01, 116.34 (d, J = 13.0 Hz), 116.63 (q, J = 5.2 Hz),

117.59, 117.74, 122.57 (q, J = 271.4 Hz), 124.98 (d, J = 8.0 Hz), 135.87, 138.65, 146.91,

152.30, 152.57, 153.98 (d, J = 249.5 Hz). mp: 223 oC

Anal Calcd for C13H9F4N3O: C, 52.18; H, 3.03; N, 14.04; Found: C, 52.40; H, 3.16; N,

13.89.

14. Synthesis of U07.

A mixture of pyridin-4-amine (1.91 g, 20 mmol) and 1,1'-Carbonyldiimidazole (CDI) (1.09 g, 6.7 mmol) in THF (25 mL) was stirred at rt for 48 h. The solvent was evaporated in vacuo. The residue was crystallized from H2O (20 mL) and dried to afford1,3-di(pyridin-

4-yl)urea (1.207 g, 84%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.45 (d, J = 6.5 Hz, 4H), 8.39 (d, J = 6.4 Hz, 4H), 9.30

(s, 2H).

13 C NMR (126 MHz, DMSO-d6) δ 112.53, 145.98, 150.26, 151.80. mp: 187-189 oC; (lit. mp 218–220 oC)19; (lit. mp 202–203 oC)20 145

Anal Calcd for C11H10N4O·0.4H2O: C, 59.67; H, 4.92; N, 25.30; Found: C, 59.53; H, 4.55;

N, 25.45.

15. Synthesis of U08.

A mixture of pyrazin-2-amine (1.045 g, 11 mmol) and 1,1'-Carbonyldiimidazole (CDI)

(810 mg, 5 mmol) in THF (19 mL) was stirred at rt 48 h. The precipitation was collected by filtration according to the method of Gube et al. (2012). The crude was crystallized from H2O (10 mL) to afford 1,3-di(pyrazin-2-yl)urea (185 mg, 17%) as a pale yellow solid.

1 H NMR (500 MHz, DMSO-d6) δ 8.34 (d, J = 2.6 Hz, 2H), 8.37 (dd, J = 2.6, 1.5 Hz, 2H),

9.14 (d, J = 1.5 Hz, 2H), 10.27 (s, 2H).

13C NMR (125 MHz, DMSO) δ 135.42, 138.82, 142.00, 148.67, 151.17. mp: 288-290 oC; (lit. mp 197 oC)21

Anal Calcd for C9H8N6O: C, 50.00; H, 3.73; N, 38.87; Found: C, 50.09; H, 3.80; N, 38.63.

16. Synthesis of U09.

To a solution of triphosgene (2.26 g, 7.6 mmol) in toluene (32 mL) at 0 oC was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (3.43 g, 19.2 mmol) in EtOAc (20 mL) 146

dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

To a solution of pyrazin-2-amine (1.8 g, 18.9 mmol) in THF (50 mL) was added 1-fluoro-

4-isocyanato-2-(trifluoromethyl)benzene (3.936 g, 19.2 mmol) in THF (20 mL) at rt. The reaction mixture was heated under reflux for 12 h. The solvent was evaporated in vacuo.

The residue was then crystallized from a mixture of CH2Cl2 (1 mL) and hexane (5 mL) and then recrystallized from diethyl ether (2 mL) to afford1-[4-fluoro-3-

(trifluoromethyl)phenyl]-3-(pyrazin-2-yl)urea (0.596 g, 11%) as a pale yellow solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.49 (t, J = 9.7 Hz, 1H), 7.67–7.79 (m, 1H), 8.04 (dd, J

= 6.5, 2.8 Hz, 1H), 8.29 (d, J = 2.7 Hz, 1H), 8.34 (dd, J = 2.7, 1.5 Hz, 1H), 9.02 (d, J =

1.5 Hz, 1H), 9.71 (s, 1H), 9.96 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 116.51 (qd, J = 32.3, 13.2 Hz), 116.72 (q, J = 4.1 Hz),

117.74 (d, J = 21.5 Hz), 122.53 (q, J = 273.5 Hz), 125.05 (d, J = 8.2 Hz), 135.28, 135.57

(d, J = 2.9 Hz), 138.11, 141.66, 149.05, 151.85, 154.13 (dq, J = 248.9, 2.3 Hz). mp: 220-222 oC

Anal Calcd for C12H8F4N4O: C, 48.01; H, 2.69; N, 18.66; Found: C, 48.19; H, 2.81; N,

18.80.

147

17. Synthesis of U10.

To a solution of triphosgene (855 mg, 2.9 mmol) in toluene (10.5 mL) at 0 oC was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (1.566 g, 8.7 mmol) in EtOAc (8 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

To a solution of pyridin-3-amine (813 mg, 8.6 mmol) in THF (26 mL) was added 1-fluoro-

4-isocyanato-2-(trifluoromethyl)benzene (1.775 g, 8.7 mmol) in THF (9 mL) at rt. The reaction mixture was stirred at 80 °C for 4 h. The solvent was evaporated in vacuo. The residue was then crystallized from EtOAc to afford1-[4-fluoro-3-(trifluoromethyl)phenyl]-

3-(pyridin-3-yl)urea (350 mg, 14%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.47 (t, J = 9.8 Hz, 1H), 7.61 (dd, J = 8.5, 5.0 Hz, 1H),

7.68 (dt, J = 9.1, 3.7 Hz, 1H), 8.00 (dd, J = 6.4, 2.8 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H),

8.35 (d, J = 5.0 Hz, 1H), 8.84 (s, 1H), 9.51 (s, 1H), 9.52 (s, 1H).

13 C NMR (126 MHz, DMSO-d6) δ 116.18 (q, J = 4.3 Hz), 116.46 (qd, J = 32.2, 13.1 Hz),

117.72 (d, J = 21.5 Hz), 122.56 (q, J = 272.0 Hz), 124.48 (d, J = 8.0 Hz), 125.47, 129.48,

135.64, 136.07 (d, J = 2.8 Hz), 137.69, 139.10, 152.52, 153.88 (dq, J = 248.4, 2.0 Hz). mp: 233-235 oC 148

Anal Calcd for C13H9F4N3O: C, 52.18; H, 3.03; N, 14.04; Found: C, 52.08; H, 3.20; N,

14.08.

18. Synthesis of U11.

To a solution of triphosgene (721 mg, 2.4 mmol) in toluene (10 mL) at 0 oC was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (1305 mg, 7.3 mmol) in EtOAc (7 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

To a solution of pyridin-4-amine (686 mg, 7.3 mmol) in THF (22 mL) was added 1-fluoro-

4-isocyanato-2-(trifluoromethyl)benzene (1496 mg, 7.3 mmol) in THF (7 mL) at rt. The reaction mixture was stirred at 80 °C for 4 h. The solvent was evaporated in vacuo. The residue was then successively crystallized from EtOAc and diethyl ether to afford1-[4- fluoro-3-(trifluoromethyl)phenyl]-3-(pyridin-4-yl)urea (235 mg, 11%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.40–7.52 (m, 3H), 7.67 (dt, J = 7.7, 3.6 Hz, 1H), 7.99

(dd, J = 6.5, 2.8 Hz, 1H), 8.38 (d, J = 6.4 Hz, 2H), 9.26 (s, 1H), 9.32 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 112.50, 116.43 (q, J = 5.2 Hz), 117.68 (d, J = 21.5 Hz),

122.54 (q, J = 272.1 Hz), 124.75 (d, J = 8.4 Hz), 135.95 (d, J = 2.8 Hz), 146.49, 149.86,

152.19, 153.94 (d, J = 249.1 Hz). 149

mp: 185 oC

Anal Calcd for C13H9F4N3O: C, 52.18; H, 3.03; N, 14.04; Found: C, 52.40; H, 3.00; N,

14.02.

19. Synthesis of U13.

To a solution of triphosgene (1.12 g, 3.8 mmol) in toluene (32 mL) at 0 oC, was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (1.79 g, 10 mmol) in EtOAc (16 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

A mixture of 3-aminobenzoic acid (2.74 g, 20 mmol) and 1-fluoro-4-isocyanato-2-

o (trifluoromethyl)benzene (2.05 g, 10 mmol) in CH3CN (40 mL) was stirred at 80 C for 22 h. After addition of 2 M HCl (200 mL) and heated at 80 oC for 10 min, the resulting precipitate was filtered and crystallized from H2O (50 mL) and then diethyl ether (10 mL) to afford 3-[3-[4-fluoro-3-(trifluoromethyl)phenyl]ureido]benzoic acid (2.718 g, 79%) as a white solid. 150

1 H NMR (500 MHz, DMSO-d6) δ 7.42 (t, J = 7.8 Hz, 1H), 7.45 (t, J = 9.9 Hz, 1H), 7.58 (d,

J = 7.7 Hz, 1H), 7.62–7.74 (m, 2H), 8.02 (dd, J = 6.5, 2.7 Hz, 1H), 8.15 (s, 1H), 9.03 (s,

1H), 9.06 (s, 1H), 12.94 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 116.15 (q, J = 4.2 Hz), 116.37 (qd, J = 32.1, 13.0 Hz),

117.56 (d, J = 21.5 Hz), 119.23, 122.61 (q, J = 272.1 Hz), 122.74, 123.01, 124.42 (d, J =

8.1 Hz), 128.98, 131.35, 136.45 (d, J = 2.7 Hz), 139.64, 152.59, 153.68 (dq, J = 248.1,

2.2 Hz), 167.23. mp: 317-320 oC

Anal Calcd for C15H10F4N2O3: C, 52.64; H, 2.95; N, 8.19; Found: C, 52.48; H, 3.11; N,

8.21.

20. Synthesis of U14.

To a solution of triphosgene (3.41 g, 11.5 mmol) in toluene (90 mL) at 0 oC, was added a solution of 4-fluoro-3-(trifluoromethyl)aniline (5.90 g, 33.0 mmol) in EtOAc (50 mL) dropwise. After stirring at 0 oC for 30 min, the reaction mixture was allowed to return to rt and then heated at 80 oC overnight. The solvents were evaporated in vacuo to afford 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene as an oil which was used for the next step without further purification.

A mixture of 4-aminobenzonitrile (1.888 g, 16 mmol) and 1-fluoro-4-isocyanato-2-

o (trifluoromethyl)benzene (1.69 g, 8.25 mmol) in CH3CN (13.75 mL) was stirred at 80 C 151

overnight. After addition of 2 M HCl (50 mL), the resulting precipitate was filtered and crystallized from diethyl ether (10 mL) to afford 1-(4-cyanophenyl)-3-[4-fluoro-3-

(trifluoromethyl)phenyl]urea (1.443 g, 54%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.46 (t, J = 9.7 Hz, 1H), 7.59–7.70 (m, 3H), 7.74 (d, J =

8.7 Hz, 2H), 7.99 (dd, J = 6.5, 2.7 Hz, 1H), 9.24 (s, 1H), 9.36 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 103.64, 116.39 (q, J = 4.9 Hz), 116.46 (qd, J = 32.3,

13.2 Hz), 117.59 (d, J = 21.5 Hz), 118.32, 119.21, 122.56 (q, J = 272.0 Hz), 124.62 (d, J

= 8.0 Hz), 133.22, 136.04, 143.89, 152.20, 153.91 (d, J = 248.3 Hz). mp: 233-235 oC

Anal Calcd for C15H9F4N3O: C, 55.73; H, 2.81; N, 13.00; Found: C, 55.95; H, 2.90; N,

13.21.

21. Synthesis of U15.

A mixture of 4-aminobenzamide (3.00 g, 22.1 mmol) and 1-fluoro-4-isocyanato-2-

o (trifluoromethyl)benzene (2.26 g, 11 mmol) in CH3CN (55 mL) was stirred at 80 C overnight. After addition of 2 M HCl (50 mL) and H2O (250 mL), the resulting precipitate was filtered and crystallized from diethyl ether (10 mL) to afford 4-[3-[4-fluoro-3-

(trifluoromethyl)phenyl]ureido]benzamide (1.87 g, 50%) as a white solid. 152

1 H NMR (500 MHz, DMSO-d6) δ 7.19 (brs, 1H), 7.45 (t, J = 9.7 Hz, 1H), 7.52 (d, J = 8.7

Hz, 2H), 7.59–7.69 (m, 1H), 7.81 (brs, 1H), 7.82 (d, J = 8.7 Hz, 2H), 8.01 (dd, J = 6.5,

2.7 Hz, 1H), 9.05 (s, 1H), 9.11 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 116.14 (q, J = 4.8 Hz), 116.41 (qd, J = 32.1, 13.0 Hz),

117.40, 117.61 (d, J = 21.4 Hz), 122.61 (q, J = 271.9 Hz), 124.43 (d, J = 8.1 Hz), 127.71,

128.51, 136.36 (d, J = 2.7 Hz), 142.14, 152.41, 153.74 (dq, J = 248.1, 2.0 Hz), 167.46. mp: 233-235 oC

Anal Calcd for C15H11F4N3O2: C, 52.79; H, 3.25; N, 12.31; Found: C, 52.65; H, 3.29; N,

12.55.

22. Synthesis of U16.

A mixture of 4-aminobenzoic acid (2.331 g, 17.0 mmol) in CH3CN (100 mL) and 1-fluoro-

4-isocyanato-2-(trifluoromethyl)benzene (1.69 g, 8.25 mmol) in CH3CN (3.75 mL) was

o stirred at 80 C overnight. After addition of 2 M HCl (50 mL) and H2O (250 mL), the resulting precipitate was filtered and crystallized from diethyl ether (60 mL) to afford 4-[3-

[4-fluoro-3-(trifluoromethyl)phenyl]ureido]benzoic acid (1.726 g, 61%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.45 (t, J = 9.8 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.61–

7.71 (m, 1H), 7.88 (d, J = 8.7 Hz, 2H), 8.01 (dd, J = 6.5, 2.7 Hz, 1H), 9.14 (s, 1H), 9.18

(s, 1H), 12.62 (s, 1H). 153

13 C NMR (125 MHz, DMSO-d6) δ 116.22 (q, J = 5.0, 4.5 Hz), 116.42 (qd, J = 32.1, 13.1

Hz), 117.53, 117.70, 122.60 (q, J = 272.4 Hz), 124.03, 124.49 (d, J = 8.0 Hz), 130.50,

136.26 (d, J = 2.8 Hz), 143.64, 152.32, 153.79 (dq, J = 248.3, 2.2 Hz), 167.02. mp: > 380 oC

Anal Calcd for C15H10F4N2O3: C, 52.64; H, 2.95; N, 8.19; Found: C, 52.88; H, 3.12; N,

8.12.

23. Synthesis of U17.

A mixture of 3-methylisoxazol-5-amine (1.078 g, 11 mmol) in CH3CN (25 mL) and 1- fluoro-4-isocyanato-2-(trifluoromethyl)benzene (1.128 g, 5.5 mmol) in CH3CN (2.5 mL) was stirred at 80 oC overnight. After addition of 2 M HCl (50 mL), the resulting precipitate was filtered and crystallized from diethyl ether (10 mL) to afford1-(4-fluoro-3-

(trifluoromethyl)phenyl)-3-(3-methylisoxazol-5-yl)urea (875 mg, 53%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 2.17 (s, 3H), 5.98 (s, 1H), 7.47 (t, J = 9.7 Hz, 1H), 7.65–

7.72 (m, 1H), 7.98 (dd, J = 6.5, 2.6 Hz, 1H), 9.20 (s, 1H), 10.31 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 11.34, 86.90, 116.45 (qd, J = 19.1 Hz), 116.68 (q, J =

32.1, 13.0 Hz), 117.68 (d, J = 21.5 Hz), 122.51 (q, J = 272.7 Hz), 124.97 (d, J = 8.1 Hz),

135.63 (d, J = 2.9 Hz), 149.92, 154.11 (dq, J = 248.8, 2.3 Hz), 160.66, 161.67. 154

mp: 180-181 oC

Anal Calcd for C12H9F4N3O2: C, 47.53; H, 2.99; N, 13.86; Found: C, 47.60; H, 3.12; N,

14.00.

24. Synthesis of U18.

A mixture of 2-aminobenzamide (1.496 g, 11 mmol) and 1-fluoro-4-isocyanato-2-

o (trifluoromethyl)benzene (1.107 g, 5.4 mmol) in CH3CN (50 mL) was stirred at 80 C overnight. After addition of 2 M HCl (200 mL), the resulting precipitate was filtered and crystallized from diethyl ether (1 mL) to afford 2-[3-[4-fluoro-3-

(trifluoromethyl)phenyl]ureido]benzamide (1.119 mg, 61%) as a white solid.

1 H NMR (500 MHz, DMSO-d6) δ 7.06 (t, J = 7.5 Hz, 1H), 7.38–7.52 (m, 2H), 7.69 (s, 1H),

7.73–7.81 (m, 2H), 8.04 (dd, J = 6.5, 2.7 Hz, 1H), 8.25 (s, 1H), 8.30 (d, J = 8.7 Hz, 1H),

10.08 (s, 1H), 10.84 (s, 1H).

13 C NMR (125 MHz, DMSO-d6) δ 116.08 (q, J = 4.8 Hz), 116.31 (qd, J = 32.2, 13.0 Hz),

117.46 (d, J = 21.3 Hz), 119.82, 120.27, 121.09, 122.65 (q, J = 271.9 Hz), 124.16 (d, J =

7.9 Hz), 128.40, 131.77, 136.90 (d, J = 2.7 Hz), 140.16, 152.37, 153.57 (dq, J = 248.0,

1.9 Hz), 170.82. mp: 242-244 oC 155

Anal Calcd for C15H11F4N3O2: C, 52.79; H, 3.25; N, 12.31; Found: C, 52.55; H, 3.45; N,

12.42.

5.6.3. Newly transformed schistosomula (NTS) drug assay22.

Cercariae were collected from S. mansoni-infected B. glabrata, mechanically transformed to schistosomula, and stored for 12 to 24 h at 37 °C and 5% CO2 in medium

199 supplemented with 5% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin before use.

For the NTS drug assay, 100 NTS per well were incubated in a 96-well plate (BD Falcon) with compounds diluted in supplemented medium 199 in 2-fold serial dilutions from 33.3 to 0.26 μM, for IC50 determinations. Wells containing NTS exposed to drug-free DMSO at a volume equivalent to the highest drug concentration in the assay (0.3%) served as controls. The efficacy of the drugs was judged by scoring their overall viability, using phenotypic reference points such as motility, morphology, and granularity.

5.6.4. Adult S. mansoni drug assay22.

Adult S. mansoni were collected from mice by dissection at 49 to 70 days postinfection and were maintained in RPMI 1640 culture medium supplemented with 5% FCS, 100

U/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2. IC50 values were assessed using 3-fold serial dilutions from 33.3 to 0.41 μM in duplicate, and this was repeated once. Compounds were diluted in supplemented RPMI medium in 24-well plates (BD Falcon), to which three worms of each sex were added per well. Worms incubated with drug-free DMSO (0.3%) served as a control. The viability of S. mansoni adult was assessed via microscopic readout (in the same manner as described for NTS). 156

5.6.5. in vivo antischistosomal screen.

As described by Keiser23, cercariae of S. mansoni were obtained from infected

Biomphalaria glabrata. NMRI mice were infected subcutaneously with approximately 100

S. mansoni cercariae. At 49 d after infection, groups of four mice were treated with single 100-400 mg/kg oral doses of compounds in a 7% (v/v) Tween 80% and 3% (v/v) ethanol vehicle (10 mL/kg). Untreated mice (n = 8) served as controls. At 21 d post- treatment, animals were killed by the CO2 method and dissected. Worms were removed by picking, then sexed and counted.

157

References:

1. McManus, D. P.; Gray, D. J.; Li, Y.; Feng, Z.; Williams, G. M.; Stewart, D.; Rey-

Ladino, J.; Ross, A. G. Schistosomiasis in the People's Republic of China: the era of the

Three Gorges Dam. Clin. Microbiol. Rev. 2010, 23, 442-466.

2. Steinmann, P.; Keiser, J.; Bos, R.; Tanner, M.; Utzinger, J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect. Dis. 2006, 6, 411-425.

3. Hotez, P. J.; Kamath, A. Neglected tropical diseases in sub-saharan Africa: review of their prevalence, distribution, and disease burden. PLoS Negl Trop Dis 2009, 3, e412.

4. McManus, D. P.; Li, Y.; Williams, G. M.; Bergquist, R.; Gray, D. J. Challenges in

Controlling and Eliminating Schistosomiasis. In Challenges in Infectious Diseases,

Springer: 2013; pp 265-299.

5. Loukas, A.; Good, M. F. Back to the future for antiparasite vaccines? Expert review of vaccines 2013, 12, 1-4.

6. Utzinger, J.; Keiser, J. Schistosomiasis and soil-transmitted helminthiasis: common drugs for treatment and control. Expert Opin. Pharmacother. 2004, 5, 263-285.

7. Dömling, A.; Khoury, K. Praziquantel and schistosomiasis. Chem. Med. Chem.

2010, 5, 1420-1434.

8. Pica-Mattoccia, L.; Doenhoff, M. J.; Valle, C.; Basso, A.; Troiani, A.-R.; Liberti, P.;

Festucci, A.; Guidi, A.; Cioli, D. Genetic analysis of decreased praziquantel sensitivity in a laboratory strain of Schistosoma mansoni. Acta Trop. 2009, 111, 82-85.

9. Gryseels, B.; Polman, K.; Clerinx, J.; Kestens, L. Human schistosomiasis. J.-

Lancet 2006, 368, 1106-1118.

10. Cioli, D.; Pica-Mattoccia, L. Praziquantel. Parasitol. Res. 2003, 90, S3-S9. 158

11. Horton, J. Human gastrointestinal helminth infections: are they now neglected diseases? Trends Parasitol. 2003, 19, 527-531.

12. Geary, T. G.; Woo, K.; McCarthy, J. S.; Mackenzie, C. D.; Horton, J.; Prichard, R.

K.; de Silva, N. R.; Olliaro, P. L.; Lazdins-Helds, J. K.; Engels, D. A. Unresolved issues in anthelmintic pharmacology for helminthiases of humans. Int. J. Parasitol. 2010, 40, 1-

13.

13. Caffrey, C. R.; Secor, W. E. Schistosomiasis: from drug deployment to drug development. Curr. Opin. Infect. Dis. 2011, 24, 410-417.

14. Sayed, A. A.; Simeonov, A.; Thomas, C. J.; Inglese, J.; Austin, C. P.; Williams, D.

L. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nat.

Med. 2008, 14, 407-412.

15. Caffrey, C. R.; Williams, D. L.; Todd, M. H.; Nelson, D. L.; Keiser, J.; Utzinger, J.

Chemotherapeutic development strategies for schistosomiasis. 2009; p 299-321.

16. Ingram-Sieber, K.; Cowan, N.; Panic, G.; Vargas, M.; Mansour, N. R.; Bickle, Q.

D.; Wells, T. N.; Spangenberg, T.; Keiser, J. Orally active antischistosomal early leads identified from the open access malaria box. PLoS Neglected Trop. Dis. 2014, 8, e2610.

17. Lee, I.-Y.; Gruber, T. D.; Samuels, A.; Yun, M.; Nam, B.; Kang, M.; Crowley, K.;

Winterroth, B.; Boshoff, H. I.; Barry, C. E. Structure-activity relationships of antitubercular salicylanilides consistent with disruption of the proton gradient via proton shuttling.

Bioorg. Med. Chem. 2013, 21, 114-126.

18. Milne, G. W. A.; Editor. Drugs: Synonyms & Properties. Ashgate Publ Co.: 2000; p 1280 pp.

19. Chandran, S. K.; Nath, N. K.; Cherukuvada, S.; Nangia, A. N–H… N (pyridyl) and

N–H… O (urea) hydrogen bonding and molecular conformation of N-aryl-N′-pyridylureas.

J. Mol. Struct. 2010, 968, 99-107. 159

20. AbdelHafez, E. S.; Aly, O. M.; Abuo‐Rahma, G. E. D. A.; King, S. B. Lossen

Rearrangements under Heck Reaction Conditions. Adv. Synth. Catal. 2014, 356, 3456-

3464.

21. Stanovnik, B.; Tisler, M.; Golob, V.; Hvala, I.; Nikolic, O. Heterocycles. CXCVIII.

Heteroacyl azides as acylating agents for aromatic or heteroaromatic amines. J.

Heterocycl. Chem. 1980, 17, 733-6.

22. Cowan, N.; Datwyler, P.; Ernst, B.; Wang, C.; Vennerstrom, J. L.; Spangenberg,

T.; Keiser, J. Activities of N,N'-diarylurea MMV665852 analogs against Schistosoma mansoni. Antimicrob. Agents Chemother. 2015, 59, 1935-1941.

23. Keiser, J.; Vargas, M.; Vennerstrom, J. L. Activity of antiandrogens against juvenile and adult Schistosoma mansoni in mice. J. Antimicrob. Chemother. 2010, 65,

1991-1995.

160

Chapter 6

Summary

Schistosomiasis is caused by parasitic trematode worms of the genus

Schistosoma in the blood vessel. The burden of disease attributable to the three major human schistosome species (Schistosoma mansoni, S. Haematobium, and S. japonicum) is estimated to be almost 200 million infected patients1. More than 90% of the roughly 200 million cases of schistosomiasis occur in Africa2, 3 and close to 800 million are at risk. It is one of the most prevalent of the tropical infectious diseases4.

In the absence of vaccines5 and with the challenges surrounding implementation of other preventive measures such as access to safe water and improved sanitation, the current and precarious mainstay for treatment and control of schistosomiasis is praziquantel (PZQ)6, 7. This dependence on a single drug is alarming, as there is some evidence that resistance to PZQ may be emerging in Africa and laboratory studies indicate that schistosomes exposed to repeated in vivo PZQ treatments have reduced drug sensitivities8, 9. Furthermore, PZQ is inactive against the juvenile schistosomula; this may be an important factor in the frequently observed treatment ‘failures’ in areas endemic for schistosomiasis. Even so, drug discovery for schistosomiasis has languished10, 11, although several antischistosomal lead compounds12, 13 have been identified in recent years.

The aryl hydantoin Ro 13-397814 was used as a starting point in this research to identify several lead compounds with high antischistosomal efficacy and with minimal to no interaction with the host androgen receptor (AR). The overall goal of this research was to elucidate the SAR of aryl hydantoins and theirs derivatives. Base on a broad library of new analogs, a new understanding of the structural specificity of antischistosomal efficacy 161

for aryl hydantoins, diaryl ureas and urea carboxylic acids was gained, to identify potential novel analogs with high antischistosomal efficacy and selectivity.

In Chapter 2, it was demonstrated that administration of 100 mg/kg oral doses of

AH01 to mice infected with adult and juvenile Schistosoma mansoni produced 95 and 64% total worm burden reductions. This result confirmed its high activity against adult worms, and demonstrated that AH01was also effective against juvenile infections. AH01 had no measureable interaction with the androgen receptor in a ligand competition assay, but it did block dihydrotestosterone-induced cell proliferation in an androgen-dependent human prostate cancer cell line. For AH01, nilutamide, and three closely related aryl hydantoin derivatives, there was no correlation between antischistosomal activity and androgen receptor interaction.

In Chapter 3, the synthesis of several aryl hydantoin analogues is described herein.

These compounds were prepared in an effort to probe the structure–activity relationship around the phenyl substituent and hydantoin core for the studies toward antischistosomal activity. Several of them, such as AR11, AR62, AR13, AR64, and AR43, showed significant antischistosomal activity and could serve as leads for further optimization.

In Chapter 4, several efficient synthetic routes were developed to make the first round of urea carboxylic acid targets. These methods can be applied to the synthesis of structurally diverse AR37 analogs. These urea carboxylic acids could lead to an advance in the structure–activity relationship of this class of compounds in antischistosomal researches.

In Chapter 5, the novel N,N'-diaryl ureas were designed and synthesized as new analogues of Ro 13-3978, some of which showed significant antischistosomal effects. The most promising compound identified in this work was N,N’-di(quinoxalin-2-yl)urea. A single

200 mg/kg oral dose of AR50 administered to S. mansoni-infected mice reduced adult 162

WBR by 75.1%, while a dose of 100 mg/kg reduced WBR by 62%. This new lead compound can serve as a new direction for further optimization. In addition, various synthetic routes to diarylureas were described. 163

Future studies 1. Perform computational chemistry studies to elucidate the interactions between aryl

hydantoin and human androgen receptor;

2. Perform immunological-based experiments to dissect the disconnection between

the low ex vivo activity and high in vivo efficacy of the aryl hydantoins. The

experiments may elucidate the antischistosomal effects that derive from host-

independent vs. host-dependent mechanisms;

3. Use affinity chromatography and tandem photoaffinity labeling-bioorthogonal

conjugation to identify potential schistosome protein targets of target compounds

with high in vitro activity.

164

References:

1. McManus, D. P.; Gray, D. J.; Li, Y.; Feng, Z.; Williams, G. M.; Stewart, D.; Rey-

Ladino, J.; Ross, A. G. Schistosomiasis in the People's Republic of China: the era of the

Three Gorges Dam. Clin. Microbiol. Rev. 2010, 23, 442-466.

3. Hotez, P. J.; Kamath, A. Neglected tropical diseases in sub-saharan Africa: review of their prevalence, distribution, and disease burden. PLoS Negl. Trop. Dis. 2009, 3, e412.

4. McManus, D. P.; Li, Y.; Williams, G. M.; Bergquist, R.; Gray, D. J. Challenges in

Controlling and Eliminating Schistosomiasis. In Challenges in Infectious Diseases,

Springer: 2013; pp 265-299.

5. Loukas, A.; Good, M. F. Back to the future for antiparasite vaccines? Expert Rev.

Vaccines 2013, 12, 1-4.

6. Utzinger, J.; Keiser, J. Schistosomiasis and soil-transmitted helminthiasis: common drugs for treatment and control. Expert Opin. Pharmacother. 2004, 5, 263-285.

7. Dömling, A.; Khoury, K. Praziquantel and schistosomiasis. Chem. Med. Chem.

2010, 5, 1420-1434.

8. Pica-Mattoccia, L.; Doenhoff, M. J.; Valle, C.; Basso, A.; Troiani, A.-R.; Liberti, P.;

Festucci, A.; Guidi, A.; Cioli, D. Genetic analysis of decreased praziquantel sensitivity in a laboratory strain of Schistosoma mansoni. Acta trop. 2009, 111, 82-85.

9. Gryseels, B.; Polman, K.; Clerinx, J.; Kestens, L. Human schistosomiasis. J.-

Lancet 2006, 368, 1106-1118.

10. Horton, J. Human gastrointestinal helminth infections: are they now neglected diseases? Trends Parasitol. 2003, 19, 527-531. 165

11. Caffrey, C. R.; Secor, W. E. Schistosomiasis: from drug deployment to drug development. Curr. Opin. Infect. Dis. 2011, 24, 410-417.

12. Sayed, A. A.; Simeonov, A.; Thomas, C. J.; Inglese, J.; Austin, C. P.; Williams, D.

L. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nat.

Med. 2008, 14, 407-412.

13. Caffrey, C. R.; Williams, D. L.; Todd, M. H.; Nelson, D. L.; Keiser, J.; Utzinger, J.

Chemotherapeutic development strategies for schistosomiasis. 2009; p 299-321.

14. Link, H.; Stohler, H. R. 3-Arylhydantoine, eine substanzklasse mit schistosomizider wirkung. Eur. J. Med. Chem. 1984, 19, 261– 265.

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