The Design and Synthesis of Cyanines and Arylimidamide Azole

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THE DESIGN AND SYNTHESIS OF CYANINES AND ARYLIMIDAMIDE AZOLE

HYBRIDS AS ANTILESIHMANAIAL AGENTS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the

Graduate School of The Ohio State University

Ahmed Abdelhameed

Graduate Program in Pharmaceutical Sciences

The Ohio State University

2018

Dissertation Committee

Professor Karl A.Werbovetz, Advisor

Professor James R. Fuchs

Professor Liva Rakotondraibe

Professor Mark J. Mitton-Fry

Ahmed Abdelhameed

2018

Abstract

Leishmaniasis is a neglected tropical disease; the number of fatalities it causes ranks it second to malaria among parasitic infections. It is found in impoverished tropical regions, making it hard to obtain funding for the development of newer antileishmanial agents. The Leishmania parasite can cause cutaneous and visceral manifestations depending on the causative parasite species.

Cutaneous leishmaniasis can lead to lifelong scars, and some forms can develop into mucocutaneous leishmaniasis, which attacks the mucosal lining of the nasopharyngeal tract.

Visceral leishmaniasis, in which the liver, spleen, and bone marrow become infected, is fatal if left untreated. Currently, antiquated antimonial drugs remain a first line antileishmanial treatment in many areas. Antimonials are given parenterally over a period of 21 days and suffer from serious toxicity issues as well as resistance on the Indian subcontinent. Paromomycin is inexpensive, yet it is given to treat visceral leishmaniasis only by the parenteral route. It also shows some toxicity issues and is given over a long treatment course. Amphotericin B deoxycholate, another injectable antileishmanial drug, is nephrotoxic. Liposomal amphotericin B mitigates toxicity issues observed with the deoxycholate formulation, yet it is very expensive especially for patients in developing countries. Finally, miltefosine has the advantage of being orally bioavailable, but this antileishmanial drug is teratogenic and already suffers from decreased efficacy when used on the Indian subcontinent.

This dissertation describes research conducted on two classes of compounds with promising antileishmanial activity. The first class was inspired by the cyanine compound thiazole orange, a lead that was originally discovered through high throughput screening of almost 200,000 compounds. It displayed potent in vitro antileishmanial activity against intracellular L. donovani amastigotes with an IC50 value of 21 nM and 66-fold selectivity towards the parasite compared to peritoneal macrophages. Thiazole orange also showed moderate in vivo efficacy with 44% reduction in liver parasitemia in L. donovani infected mice at intraperitoneal dose of 1 mg/kg/day for five days. The second class of compounds possesses the arylimidamide scaffold. Lead arylimidamide DB766, a compound synthesized by the Boykin lab at Georgia State University, displayed very potent antileishmanial activity when tested in our lab with an IC50 value of 36 nM in L. donovani intracellular macrophage assay and good selectivity index towards the parasite compared to the J774 macrophages (CC50 value of 2.8 µM). Oral administration of DB766 led only to 71% reduction in liver parasitemia in L. donovani infected mice at a dose of 100 mg/kg for 5 days which resulted in the discontinuation of the preclinical investigation of this compound.

The potent in vitro antileishmanial activity and encouraging in vivo efficacy of these two compounds partially inspired the synthesis of the two classes of molecules described in this dissertation in the hopes of identifying new antileishmanial drug candidates or improved leads.

Chapter 1 provides background information about leishmaniasis, the parasite life cycle, disease epidemiology and clinical manifestations of the disease. This chapter also reviews the current antileishmanial agents as well as candidate compounds in research and in preclinical trials.

Finally, a brief background about the antileishmanial activity of previous cyanines and arylimidamides is presented.

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Chapter 2 provides the methodology on which the synthesis of cyanine analogs was based followed by the synthetic schemes for the cyanines investigated here. A brief discussion about the conformation of these cyanines is provided, which issupported by the X-ray crystallography study of 2.1q conducted by Dr. Carla Slebodnick at Virginia Tech. The in vitro antileishmanial assays and J774 macrophage toxicity studies conducted by Xiaoping Liao, Kelsey Bryant and

Dr. April Joice will also be discussed. Briefly, the cyanines 2.1b-t displayed antileishmanial potency which differs little from the parent compound, while ring disjunction analogs 2.1u-w were less potent as well as less toxic than 2.1a-t. Finally, DNA melting studies conducted by the

Wilson lab at Georgia State University carried out with selected analogs revealed no significant change in DNA melting temperature regarding either the position or type of the substituent on the quinoline ring moiety, while ring disjunction analogs showed lesser effects on DNA melting.

Chapter 3 will focus on arylimidamide azole hybrids. Firstly, the approach for the design of the the analogs will be discussed followed by the synthetic schemes for both phenoxyalkyl and biphenoxyalkyl analogs. The in vitro antileishmanial activity, cytotoxicity, and in vivo efficacy of these analogs, which was determined by Dr. April Joice, Xiaoping Liao, Emilia Zywot, Heidi

Meeds, and Yena Kim, will also be presented. Regarding the phenoxyalkyl analogs, AIA azole analog 3.27c possessing a phenoxyoctyl linker and an isopropoxy group ortho to the imidamide group displayed the best antileishmanial activity, possessing an IC50 value of 0.57 µM against L. donovani intracellular amastigotes. Most of the biphenoxy alkyl analogs displayed potency in the submicromolar range. Compound 3.33j was the most potent, exhibiting an IC50 value of 0.15 µM against intracellar L. donovani amastigotes. In vivo evaluation of 3.27c showed that the compound led only to 33% reduction of liver parasitemia in L. donovani-infected mice upon

iv treatment orally with a dose of 75 mg/kg/day for five days. Finally, a brief discussion concerning the metabolic stability of selected compounds conducted by Dr. Michael Wang’s lab at the

University of Kansas will be given.

Chapters 4 and 5 will provide the detailed chemical characterizations of cyanines and arylimidamide azole hybrids, while the NMR spectra of the target compounds appear in the appendix.

Dedication

To my wife Walaa, my two beautiful daughters Lily and Layan and my family back in Egypt, I

dedicate this thesis

Acknowldegments

Firstly, I have to deeply thank my advisor Dr. Karl Werbovetz for allowing me to work in his lab and giving me project in areas I have no previous experience with. Also, while mentoring me he was very patient with me and was always offering me constructive advices and suggestions which greatly influenced my scientific skills and knowledge. Also, Dr Werbovetz helped me to be a better medicinal chemist by pushing me to get better understanding of the biological aspect and not only chemistry. Beside his scientific supervision me he was very helpful and welcoming whenever I faced any problems during my stay at OSU which greatly influenced my scientific progress in the lab. Secondly, I am really grateful to my committee members Dr. Fuchs, Dr.

Mitton-Fry and Dr. Liva for offering me suggestions and advice regarding my synthesis, interpretation of spectral data, and helping me with purification issues of my compounds. Dr

Mitton-Fry allowed me to use some of his lab tools and I am very thankful for that. Also, Dr.

Liva was a great help in obtaining a crystal structure for one of our compounds. Also I am thankful to my previous committee member Dr. Li for his help.

Also, I would like to thank all current and previous members in our lab for their help especially

Dr April Joice, Xioaping Liao, Emilia Zywot, Heidi Meeds and Yena Kim for conducting the biological experiments in our lab. Also, I am thankful to Dr Xiaohua Zuo, Dr Sihui Long and

Julain Richard for their help during my first year at OSU and Kelsy Bryant for help during my

vii last year of my stay in OSU. Also I would like to thank Dr. Wilson and his postdoc Dr. Guo at

GSU for performing the DNA binding studies of our compounds in his lab. Also I am thankful to

Dr Boykin and his group members particulary Dr. Farahat for their support regarding the arylimidamide project. I am also thankful to Dr. Slebodnick at Virginia Tech for performing x- ray crystallography studies on one of the cyanine compounds in our lab. I am really indebted to

Dr. McElroy the director of instrumentation in our College for providing me the training for all needed instrumentation. Also Dr. McElroy was very helpful and knowledgable whenever I asked him a question and he even developed the HPLC method for detection of the purity of cyanine analogs. I also have to thank Dr. Krishnan for her help in maintaining the instrumentation as well as the Fuchs and Mitton-Fry group members especially Dr. Sandip Vibhute for their help and sharing of their chemistry knowledge with our lab.

I am obliged to thank our previous dean Dr. Brueggemeier and Mrs Brueggemeier for establishing the Brueggemeier fellowship which I was awarded. I also thank the Egyptian government for supporting me for my first four years at OSU and the United States Department of Defense for funding me during the rest of my stay in the USA through a grant.

I am really grateful to my mother Ayda, my late father Muhamed, my aunt Hoda and my sister

Eman for their endless love, support and sacrifices. Finally, I have to thank my beloved wife who was a constant support and helped me selflessly to succeed and to pursue my career in the United

States.

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Vita

2005...... B. Pharmacy, Helwan University, Egypt

2012...... M.S. in Pharmaceutical Chemistry, Helwan University, Egypt

Fields of study

Major Field: Pharmacy

Table of Contents

Abstract ...... ii

Dedication ...... vi

Acknowledgments...... vii

Vita ...... ix

List of Tables ...... xiv

List of Figures ...... xv

List of Schemes ...... xvii

Chapter 1: Introduction ...... 1

1.1 Socioeconomic burden of leishmaniasis ...... 2

1.2 Leishmania life cycle ...... 3

1.3 Global distribution and classifications of leishmaniasis ...... 6

1.3.1 Cutaneous leishmaniasis ...... 6

1.3.2 Visceral leishmaniasis ...... 8

1.4 Leishmaniasis control method ...... 10

1.5 Current antileishmanial agents...... 11

1.5.1 Antimonial drugs ...... 11

1.5.2 Pentamidine...... 13

1.5.3 Amphotericin B ...... 13

1.5.4 Paromomycin ...... 15

1.5.5 Azoles ...... 16

1.5.6 Miltefosine ...... 17

1.5.7 Sitamiquine ...... 18

1.5.8 Drug combinations ...... 18

1.6 Promising antileishmanial leads ...... 20

1.6.1 Proteasome inhibitors...... 20

1.6.2 Aminopyrazoles ...... 21

1.6.3 Nitroimidazoles ...... 23

1.7 Introduction to compounds under current investigation ...... 24

1.7.1 Cyanines ...... 24

1.7.2 Arylimidamides...... 29

1.7.2.1 Investigation of the anileishmanial mechanism of arylimidamide

DB766 ...... 35

Chapter 2: Design and synthesis of cyanines as antileishmanial agents ...... 37

2.1 Aim of the work ...... 37

2.2 Chemistry ...... 39

2.3 Crystal structure of 2.1q ...... 47

2.4 Biological data ...... 48

2.5 DNA binding studies...... 52

2.6 Cyanine mechanism of action ...... 54

Chapter 3: Design and synthesis of arylimidamide azole hybrids as antileishmanial agents ...... 55

3.1 Aim of the work ...... 55

3.2 Chemistry ...... 62

3.3 Biological data ...... 74

3.3.1 In vitro antileishmanial activity ...... 74

3.3.2 In vitro metabolic stability studies ...... 82

3.3.3 In vivo efficacy of 3.27c ...... 84

3.4 Conclusion ...... 85

Chapter 4: Experimental section ...... 89

Materials and Methods ...... 89

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4.1 Cyanines ...... 91

4.2 Arylimidamide azole hybrids ...... 130

4.3 Biological data ...... 223

References ...... 227

Appendix: NMR spectra of cyanines and arylimidamide azole hybrids ...... 258

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List of Tables

Table 2.1 In vitro potency of 2.1a-p against L. donovani axenic amastigotes and J774 macrophages ...... 50

Table 2.2 In vitro potency of 2.1q-w against L. donovani axenic amastigotes and J774 macrophages ...... 51

Table 2.3 DNA binding of selected cyanines to synthetic double stranded oligomers ...... 53

Table 3.1 In vitro antileishmanial activity and toxicity of 3.12a-l ...... 76

Table 3.2 In vitro antileishmanial activity and toxicity of 3.12m, n, 3.20a-c and 3.27a-c ...... 77

Table 3.3 In vitro antileishmanial activity and toxicity of 3.33a-j ...... 80

Table 3.4 In vitro antileishmanial activity and toxicity of 3.42a-d and 3.51a-f ...... 81

Table 3.5 Metabolic stability of selected AIA azole hybids ...... 84

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List of Figures

Figure 1.1. Life cycle of Leishmania ...... 5

Figure 1.2 Geographical distribution of cutaneous leishmaniasis ...... 6

Figure 1.3 Cutaneous and mucocutaneous leishmaniasis ...... 7

Figure 1.4 Geographical distribution of visceral leishmaniasis ...... 8

Figure 1.5 Visceral leishmaniasis cases in Bihar, India ...... 9

Figure 1.6 Past and current antileishmanial drugs and clinical candidates...... 19

Figure 1.7 Structure of GNF6702 ...... 21

Figure 1.8 Structure of investigated aminopyrazoles ...... 22

Figure 1.9 Structure of nitroaromatic antileishmanial candidates ...... 24

Figure 1.10 Structure of previously tested cyanines ...... 26

Figure 1.11 Structure of cyanines discovered by high throughput screening ...... 28

Figure 1.12 Structures of pentamidine and previously synthesized imidamide analogs ...... 30

Figure 1.13 Modifications performed on the lead compound DB766 ...... 33

Figure 2.1 Proposed cyanine analogs...... 39

Figure 2.2 X-ray crystal structure of 2.1q...... 47

Figure 2.3 Circular dichroism titration spectra of 2.1f ...... 53

Figure 3.1 In vivo efficacy of DB766-posaconazole combinations ...... 57

Figure 3.2 Examples of hybrid analogs with antileishmanial activity, along with some compounds that inspired their synthesis ...... 59

Figure 3.3 Hybrid synthetic strategy ...... 61

Figure 3.4 SAR studies of AIA azole hybrids ...... 82

Figure 3.5 In vivo efficacy of 3.27c ...... 85

Figure 3.6 Proposed structural modifications ...... 88

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List of Schemes

Scheme 2.1 Synthesis of 2.6a-j...... 40

Scheme 2.2 Synthesis of 2.6k,l ...... 41

Scheme 2.3 Synthesis of 2.12a-c ...... 42

Scheme 2.4 Synthesis of 2.12d ...... 43

Scheme 2.5 Synthesis of 2.16a-d ...... 44

Scheme 2.6 Synthesis of 2.1a-t...... 45

Scheme 2.7 Synthesis of 2.1u ...... 46

Scheme 2.8 Synthesis of 2.1v,w ...... 46

Scheme 3.1 Synthesis of 3.12a-j...... 63

Scheme 3.2 Synthesis of 3.12k,l ...... 64

Scheme 3.3 Synthesis of 3.12m,n ...... 65

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Scheme 3.4 Synthesis of 3.20a-c ...... 66

Scheme 3.5 Synthesis of 3.27a-c ...... 67

Scheme 3.6 Synthesis of 3.33a-j...... 69

Scheme 3.7 Synthesis of 3.42a-d ...... 71

Scheme 3.8 Synthesis of 3.51a-f...... 73

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Chapter 1

Introduction

Leishmaniasis is a tropical vector-borne disease endemic in 98 countries worldwide caused by 20 different Leishmania species [1]. It is acquired through the bite of the female sandfly of the genus Phlebotomus for Old World leishmaniasis and the genus Lutzomyia for New World leishmaniasis. It is considered one of the most dangerous neglected infectious diseases as the number of fatalities rank it second only to malaria. The disease manifests itself as either cutaneous leishmaniasis or visceral leishmaniasis. There are almost 0.2-0.4 million visceral leishmaniasis cases annually with approximately triple that number for the cutaneous form [2].

Since leishmaniasis is mostly present in impoverished regions, this increases the risk of infections in these communities with almost 350 million people at risk of both forms of leishmaniasis according to the World Health Organization (WHO) [3]. Leishmania infection is endemic in tropical regions and can result in visceral or cutaneous leishmaniasis. Visceral leishmaniasis is most often caused by L. donovani and L. infantum. Cutaneous leishmaniasis can be further classified into Old World leishmaniasis which is endemic in Africa, the Middle East and the Indian subcontinent and is caused by L. tropica, L. major, L. aethiopica or sometimes L. donovani or L. infantum. New World leishmaniasis is caused by species of the L. mexicana

1 complex (L. mexicana, L. amazonensis, and L. venezuelensis) and members of the Viannia subgenus (including L. braziliensis, L. guyanensis, and L. panamensis) [4].

1.1 Socioeconomic burden of leishmaniasis

Leishmaniasis is mostly found in developing countries; this puts patients from these areas under both economic and social burdens. Also, it affects the welfare of the society by decreasing the productivity of the labor force and having a negative impact on the economy. For example, the visceral leishmaniasis treatment course costs around 425 US dollars per person in Nepal which is more than the average family in Nepal spends on healthcare in one year in this developing country [5]. A similar study conducted in India showed that patients in the poor areas of this country spend around 11% of their household income annually for a course of visceral leishmaniasis treatment [6]. As mentioned earlier, leishmaniasis is present in poor regions where sanitation, pest control and medical treatment are substandard compared to developed countries, making the control of the disease and eradication of the vector sandflies much harder. Another study done in Afghanistan showed than only 22 percent of leishmaniasis patients in that country are able to afford insecticide-treated bed nets required to limit sandfly bites, demonstrating how poverty might contribute indirectly to the spread of the disease [7].

Another aspect of the disease that is rarely discussed is its psychological effect on leishmaniasis patients. Lack of education in many of these impoverished societies fosters the belief that cutaneous leishmaniasis patients with disfiguring scars on their bodies are contagious. As a consequence, such patients become isolated from their societies, affecting in particular women and children attending schools in these areas [8]

Leishmaniasis is also a threat outside tropical and developing countries. Some cases of the disease have been reported in the United States of America, Canada and European countries due to globalization, military operations, a surge in international travelling and imported animals [9-

12]. This has led to an increased number of individuals at risk of contracting these parasitic disease worldwide, especially immunocompromised individuals, pregnant women and children.

1.2 Leishmania life cycle

Leishmania belongs to the family Kinetoplastida and is therefore similar to all other

Trypanosomatid parasites. This parasite has 2 different morphological life stages, the promastigote form and the human infective amastigote form, which are present in the insect vector or the mammalian host, respectively. Promastigotes have an elongated cellular body with a flagellum approximately the same length as that of the parasite. This flagellum increases the motility of the parasite, helping it to move beyond the skin collagen matrix [13]. This raises some questions concerning the pathogenesis of both cutaneous and visceral forms of the disease.

Phagocytosis of the parasite could occur by the recruited macrophages at the site of the insect bite or could occur at other locations [14]. On the other hand, amastigotes have spherical bodies with a small non motile flagellum, reducing the parasite surface area exposed to the drastic environment of parasitophorous vacuole within the host macrophages. The flagellum may also assess macrophage health, determining whether the amastigote should or shouldn’t divide within the macrophage [14].

The life cycle of Leishmania (shown in Figure 1.1) starts when the sandfly transfers Leishmania promastigote forms to the host during a blood meal. Promastigotes are eventually taken up by

3 the host macrophages and differentiate into the amastigotes in the parasitophorous vacuoles.

There are two different forms of the parasitophorous vacuoles depending on the type of the

Leishmania amastigotes. Type I parasitophorous vacuoles, as in the case of L. amazonensis infection, allow only the presence of multiple amastigotes within the same vacuole. Type II parasitophorous vacuoles, as in the case of L. major, allow only the presence of one amastigote per vacuole [15]. After multiplying in the macrophages of several different types of tissues, amastigotes cause macrophage lysis and go on to infect other cells. The amastigote form is transferred to the vector sandfly during another blood meal. After the ingestion of infected macrophages, the amastigote form differentiates to the promastigote form within the insect midgut, where the pH and temperature are different from the acidic macrophage environment.

The parasite divides in the midgut then moves after 4-5 days to the sandfly esophagus and salivary gland, where the cycle is repeated once more [16].

Figure 1.1: Life cycle of Leishmania

Picture adapted from CDC website [16]

1.3 Global distribution and classifications of leishmaniasis

1.3.1 Cutaneous leishmaniasis

Figure 1.2: Geographical distribution of cutaneous leishmaniasis

Adapted from WHO website [17]

Most cutaneous leishmaniasis cases are found in Brazil, Colombia, and Peru on the South

American continent, in Algeria in sub-Saharan Africa, and in Afghanistan, Iran, Iraq, Pakistan and Syria on the Asian continent with more than 5000 cases reported in each of these countries according to WHO in 2015. There are many other cases reported worldwide according to WHO

6 as shown in Figure 1.2 [17]. The symptoms of cutaneous leishmaniasis include elevated body temperature and swelling at the site of the insect bite. The disease progresses with development of papules that turn to nodules which ulcerate over time in case of Old World cutaneous leishmaniasis as shown in Figure 1.3A [18, 19]. Sometimes Old World leishmaniasis can develop into hyperkeratotic like lesions as shown in Figure 1.3B [19]. However, in New World cutaneous leishmaniasis, ulcerative lesions are more common as shown in Figure 1.3C.

Cutaneous leishmaniasis can self-heal over time, but permanent scarring is frequently the result.

However 1-10% of the New World leishmaniasis infections can develop into a more serious form of the disease which is termed mucocutaneous leishmaniasis. Mucocutaneous leishmaniasis is mainly caused by L. braziliensis, L. guyanensis, and L. panamensis. It damages the mucosal lining of the naso-oropharyngeal airway and it can cause serious disfiguration of the face as shown in Figure 1.3E; in some cases mucocutaneous leishmaniasis is life threatening [18, 19].

1.3A 1.3B 1.3C 1.3D 1.3E

Figure 1.3: Cutaneous and mucocutaneous leishmaniasis [reprinted with the permission from Elsevier Publishing group: Lancet [19]

1.3.2 Visceral leishmaniasis

Figure 1.4: Geographical distribution of visceral leishmaniasis

Adapted from WHO website [17]

More than 90% of visceral leishmaniasis cases are found in 8 countries which are Brazil in South

America, Ethiopia, Kenya, Somalia and Sudan in Africa, and China, India, and Nepal in Asia as reported by WHO in 2015 (Figure 1.4) [17]. Visceral leishmaniasis is caused by L. donovani in

India and East Africa, L. infantum in the Mediterranean region, and L. infantum (synonymously named L. chagasi) in South America. On rare occasions, viscerotropic leishmaniasis can occur after infection by L. tropica or L. amazonensis. Onset of the disease symptoms might take a few months or the infection can remain asymptomatic and appear once the patient is immunocompromised. The parasite attacks internal organs, primarily the spleen, liver or the bone marrow and it can be life threatening if left untreated. The symptoms include fever, anemia, hepatosplenomegaly and weight loss as shown in Figure 1.5A. Relapse can occur in some fully treated patients. This condition, called post kala-azar dermal leishmaniasis (PKDL), is characterized by hypo pigmented papular-nodular lesions on the face or the chest. Such lesions can heal spontaneously or could persist for several years, requiring additional treatment [18, 19].

1.5A 1.5B

Figure 1.5: Visceral leishmaniasis cases in Bihar, India [reprinted with the permission from Elsevier Publishing group: Lancet[19]

1.4 Leishmaniasis control methods

Aside from chemotherapy as a means to address both forms of leishmaniasis, control methods should be employed in order to decrease the spread of the disease in its endemic regions. These methods include the use of vector and reservoir control and progress in the development of antileishmanial vaccines. The use of insecticide treated materials as bed nets and domiciliary spraying with insecticides are considered the conventional sandfly control methods. The use of bed nets alone have mixed efficacy for the control of both cutaneous and visceral leishmaniasis

[20, 21]. The combination of bed nets and in house spraying or culling of infected animals led to positive effects for both forms of the disease [22, 23]. Regarding vaccines, the number of antileishmanial vaccine studies has increased over recent years due to greater knowledge of the cell mediated immune response towards the parasite. Such vaccines can be classified into three major groups: live attenuated parasite vaccines, killed parasite vaccines and defined vaccines.

First generation Leishmania vaccines consist of killed and live attenuated vaccines while the second generation vaccines include recombinant DNA and antigen containing vaccines. Whole cell killed parasite vaccines are more favored than live attenuated vaccines since they are less expensive and there are no fears that the parasite might revert back to a virulent form, especially in immunocompromised patients. However, whole cell vaccines do not cause the same immunogenic response as natural infection [24]. First generation vaccines have been studied more extensively in human and animal models yet they show serious weaknesses. The final vaccine category, defined vaccines, includes recombinant vaccines, DNA vaccines, antigen cocktail vaccines, and chimeric vaccines. Recombinant vaccines are antigenic proteins made by genetically engineered cells [25]. There are also antigen cocktail vaccines which are a group of

10 defined antigens encapsulated in cationic liposomes that showed promising protective results in canine visceral leishmaniasis models [26]. Second generation vaccines have been studied only in animal models, as insufficient data are available concerning side effects, pharmaceutical companies have provided little funding for this work, and the diversity of Leishmania strains makes it hard to successfully develop a human vaccine.

1.5 Current antileishmanial agents

1.5.1 Antimonial drugs

The history of antimonial drugs goes back to the early 20th century when trivalent antimonials were used for the treatment of leishmaniasis in India [27]. However, these drugs exhibited profound toxicity and further use was no longer recommended by the mid-1940s when they were replaced by the pentavalent antimonials [28-30]. The pentavalent antimonials are prodrugs which release the active trivalent form within the host. There are two current formulationsas shown in

Figure 1.6 used which are sodium stibogluconate (Pentostam) (1.1) made from stibonic acid and gluconic acid or meglumine antimonite (Glucantime) (1.2) which is made from pentavalent antimony and N-methyl-D-glucamine. Antimonial drugs are used for both visceral and cutaneous leishmaniasis treatment in injection formulations only as they have poor oral bioavailability [31].

In the case of visceral leishmaniasis, pentavalent antimonials are used for L. donovani leishmaniasis in Africa or L. infantum leishmaniasis in Mediterranean countries with more than

90% cure rate over a 30 day treatment course [32, 33]. They aren’t considered the first line of treatment, however, as WHO recommends either drug combinations or liposomal amphotericin

B (AmBisome) as the first line of treatment. However, in leishmaniasis caused by L. infantum,

11 pentavalent antimonials are the first line of treatment for mild and moderate cases [34].

Antimonial drugs display resistance issues in Bihar, India and though they are still active in neighboring regions, WHO doesn’t recommend their use there [35]. Antimonials are also used for cutaneous leishmaniasis with cure rate of 85-90% for Old World leishmaniasis and 26-100% for New World leishmaniasis depending on the region and the causative parasite species [36].

Resistance to antimonials is partially attributed to the misuse of the drugs where it was found that only 26% followed the WHO recommended treatment protocol (20 mg/kg for 20 or 40 days in new and relapsed cases, respectively) [37]. Also, there is another postulate that exposure of residents of Bihar to trivalent arsenic compounds present in drinking water led to cross resistance to sodium stibogluconate [38]. It was found that the concentration of antimonial drugs is lower in resistant parasites compared to antimony sensitive parasites [39]. Factors that may contribute to resistance development include inhibition of the conversion of the pentavalent prodrug form to the active trivalent species as well as decreased internalization of the drug or the increase in efflux by the parasite [40]. Overexpression of some transporter proteins such as ABC binding cassette transporters enhance efflux and lead to sub therapeutic accumulation of antimonials in the phagolysosome [41]. Both trypanothione, a bis-glutathionyl spermidine molecule found in kinetoplastid parasites, as well as multi drug resistance protein A (MRPA) transporter overexpression might play a role in resistance to antimonial drugs. The trypanothione metal complex is sequestered into cellular organelles by the aid of the MRPA transporter or is forced out of the cell by other efflux mechanisms [42]. Many mutant strains resistant to antimonials have shown increased production of trypanothione and other reduced thiols such as cysteine and glutathione which might decrease the oxidative stress induced by the antimonial drugs on the

12 parasite [43]. In terms of toxicity, pentavalent antimonials can cause pancreatitis which might be fatal, especially in immunocompromised patients. Hepatotoxicity, nephrotoxicity, and peripheral neuropathy are other concerns, since these drugs contain heavy metals that might be deposited in the liver or spleen after long treatment courses. Pentavalent antimonials can also cause arrhythmia, possibly leading to cardiac arrest [34, 44].

1.5.2 Pentamidine

Pentamidine (1.3) as shown in Figure 1.6 is an antimicrobial compound bearing bis phenylamidino groups which was used as a second line treatment for visceral leishmaniasis in

India in the form of intravenous or intramuscular injections. However, safety issues and rapid resistance development led to its discontinuation in India by the 1990s [45-47]. Pentamidine can cause nephrotoxicity, myocarditis, and hypoglycemia which is very dangerous especially in diabetic patients [45, 46]. However, pentamidine is still used intramuscularly as the isethionate salt in the treatment of cutaneous leishmaniasis cases in South America [48]. The mechanism of resistance to pentamidine is unknown but it is postulated that it is caused by the pentamidine resistance protein 1 (PRP1) transporter that belongs to the ABC transporter family [49]. It is reported that pentamidine use can lead to cross resistance to antimonial drugs [50].

1.5.3 Amphotericin B

Amphotericin B (1.4) as shown in Figure 1.6 is a macrolide polyene antibiotic which is considered a first line treatment, especially where antimonial resistance is present. Amphotericin

B binds strongly to ergosterol and the resulting complex leads to pore formation in the

13 leishmanial cell membrane and leakage of cellular contents as ions and small metabolites which finally lead to cell death [51-53]. Amphotericin B is present in two formulations and both are applied as intravenous infusions. The amphiphilic nature of amphotericin B prevents its use via the oral route, though studies showed that an oral amphotericin formulation had promising results in L. donovani infected mice [54]. Currently there are 2 parenteral amphotericin formulations: amphotericin deoxycholate (Fungizone) and amphotericin B liposomal formulation

(AmBisome). Amphotericin B deoxycholate is cheaper however it requires a long treatment course which spans around 28 days and displays more adverse side effects such as nephrotoxicity, hypokalemia and cardiac toxicity [55, 56]. AmBisome is a less toxic liposomal amphotericin B formulation that decreases the direct exposure of amphotericin B to body organs.

Also, liposomal formulations result in the fast accumulation of the drug in the liver and the spleen, allowing larger and safer doses to be administered over a short period of time [57-59].

The major drawback of AmBisome is the cost of treatment, which is considered a huge burden for patients in developing countries [57].

Amphotericin B can be used for both visceral and cutaneous leishmaniasis. For visceral leishmaniasis in India, amphotericin B deoxycholate formulation has an excellent cure rate of approximately 100% in both antimony resistant and sensitive L. donovani strains at an administered dose ranging between 7-20 mg/kg as an intravenous infusion [34]. AmBisome shows comparable results where a single dose of 10 mg/kg leads to a 95.7% cure rate [60]. In the

Mediterranean region, AmBisome provided an almost 100% cure rate after total doses up to 20 mg/kg [61]. For African L. donovani species, cure rates higher than 90% are observed for the deoxycholate formulation when administered on alternating days at 1 mg/kg for 30 days [62].

AmBisome administered at a dose of 30 mg/kg for 6 days displayed a 92.4% cure rate in

Sudanese visceral leishmaniasis [63]. Both formulations can be used for cutaneous and mucocutaneous leishmaniasis with a dose regimen of 0.7 mg/kg for the deoxycholate formulation as an intravenous infusion for 30 days or a total infusion dose up to 20-40 mg/kg for the liposomal formulation [64-66].

1.5.4 Paromomycin

Paromomycin (1.5) as shown in Figure 1.6 is an aminoglycoside antibiotic targeting ribosomal protein synthesis [67]. It has been used to treat both visceral and cutaneous leishmaniasis in either parenteral or ointment formulations. In the case of visceral leishmaniasis, a phase III clinical study conducted in Bihar showed that paromomycin had a comparable cure rate to amphotericin B with more than 94.6% of patients fully cured [68]. However, an African visceral leishmaniasis study showed that monotherapy with paromomycin at a dose of 15 mg/kg for 21 days has a very low cure rate (14.3 and 46.7% in two Sudanese centers) [69]. There are no reported studies concerning the use of paromomycin for Mediterranean and American leishmaniasis cases [70]. Topical application of paromomycin can be used for uncomplicated

Old World cutaneous leishmaniasis. A New World cutaneous leishmaniasis topical treatment study caused by L. mexicana, L. panamensis, and L. braziliensis in Ecuador and Guatemala led to 70-90% cure rate, limiting the use of paromomycin ointment there [71, 72]. The main advantage of paromomycin is its affordability where the treatment course might not exceed 10 dollars, yet it has some side effects like other aminoglycoside antibiotics (reversible ototoxicity and nephrotoxicity) when given systemically [68].

1.5.5 Azoles

Azoles are antifungal agents that work by blocking the synthesis of ergosterol by the inhibition of the 14α demethylase (CYP51) enzyme. Ergosterol is an important component in the leishmanial cell membrane, Studies have been conducted with the azole antifungal agents as shown in Figure 1.6 ketoconazole (1.6), itraconazole (1.7), and fluconazole (1.8) for their use for the treatment of cutaneous leishmaniasis with variable efficacy depending on the causative parasite species. Oral administration of itraconazole and fluconazole led to low cure rates in studies conducted on L. major infections in Indian and Mediterranean regions and among travelers [73, 74]. However, higher oral doses of fluconazole (8 mg/kg) showed a 100% cure rate for studies conducted on New World cutaneous leishmaniasis due to L. braziliensis [75].

Teatment of kala-azar leishmaniasis with escalating oral fluconazole of 20 patients in India led to apparent cure of 11 of the cases. However, the treated cases relapsed again and needed antimony therapy. Fluconazole might not have provided full protection at the tested dose, or it may not have helped the host to develop the needed immunologic defense to prevent relapse [76]. Oral administration studies with ketoconazole led to 76% cure rates for cutaneous leishmaniasis caused by L. braziliensis [77]. Administration of ketoconazole for Old World cutaneous leishmaniasis showed 80% and 89% cure rates in Kuwait and Iran respectively, however no promising effect was found on cutaneous leishmaniasis cases in Turkey [78-80]. In a study conducted by Sundar et al. on antimony resistant visceral leishmaniasis patients in India using ketoconazole at an oral dose of 200 mg/kg twice daily for 15 days, promising antileishmanial activity was not observed and the study was discontinued before its completion [81]. The

16 combination of ketoconazole and Pentostam showed a 92.3% cure rate against cutaneous leishmaniasis in studies conducted in Egypt [82].

1.5.6 Miltefosine

Miltefosine (1.9) as shown in Figure 1.6 is a phosphocholine analog synthesized originally for anticancer applications; by the end of the last century many laboratories had demonstrated the antileishmanial activity of this compound [83]. It was finally approved as an antilieshmanial drug in India by 2002, and two years later it entered the German market to be used especially in immunocompromised patients. A phase II clinical trial with miltefosine showed a 94% cure rate in HIV negative visceral leishmaniasis patients in Ethiopia [84]. Miltefosine is the first orally administered drug approved for the treatment of visceral leishmaniasis on the Indian subcontinent. Miltefosine’s mechanism of action is not fully understood. It is postulated that it interferes with phospholipid biosynthesis and alkyl lipid metabolism. [85]. A decrease in the therapeutic response to miltefosine has been observed in India and Nepal with 10% and 20% relapse rates, respectively [86, 87]. Miltefosine has a long elimination half-life of around 120 hours [88]; this leads to the exposure of patients to sub therapeutic levels for some weeks after successful treatment, possibly leading to resistance in areas where miltefosine is extensively used. Miltefosine internalization by resistant parasites is less than in miltefosine sensitive parasites due to possible reduced uptake or increased efflux of miltefosine by overexpression of

ABC transporters [87-89]. Like other antileishmanial drugs, miltefosine has some side effects that include gastrointestinal disturbances and teratogenicity [86].

1.5.7 Sitamaquine

Sitamaquine (1.10) as shown in Figure 1.6 is an antileishmanial orally active quinoline containing compound which is structurally similar to primaquine. Sitamaquine has a short half- life which prevents rapid development of resistance [90]. Sitamaquine showed a 100% cure rate for visceral leishmaniasis cases in India at doses of 2 mg/kg/day for 28 days [91]. The same dose regimen used to treat visceral leishmaniasis cases in Kenya led to only an 80% cure rate; higher doses are needed to obtain better biological activity [92]. Unfortunately, sitamiquine has a narrow therapeutic index as doses higher than 2 mg/kg are nephrotoxic and lead to methemoglobinemia [90].

1.5.8 Drug combinations

Due to the limited number of antileishmanial drugs as well as the fast rise of resistance to such agents, combinations incorporating two or more drugs working by different mechanisms are increasingly used. Pharmacokinetic properties play a role in the choice of drug combinations; a potent and short elimination half-life drug is typically used with a long half-life drug to eliminate remaining parasites as in artemisinin combination therapy for malaria. The use of drug combinations reduces the chances of the development of resistance, allows the administration of drugs at lower doses and consequently decreases their side effects. Sodium stibogluconate and paromomycin led to an 88% cure rate in antimony resistant leishmaniasis cases in Bihar [93, 94].

The same drug combination in Africa showed excellent results with more than a 97% cure rate

[95]. Single dose AmBisome followed by miltefosine treatment for a 10 day course showed an outstanding 98% cure rate in a study conducted in Bihar. These studies have shown that

18 amphotericin B, miltefosine and paromomycin can be used in combination therapy for visceral leishmaniasis [86].

Figure 1.6: Past and current antileishmanial drugs and clinical candidates

1.6 Promising antileishmanial leads

1.6.1 Proteasome inhibitors

GNF6702 (1.11) as shown in Figure 1.7, a triazolopyrimidine compound discovered through high throughput screening of approximately three million compounds, displays antiparasitic activity against the three main neglected tropical diseases leishmaniasis, Chagas disease and human African trypanosomiasis [96]. GNF6702 shows good activity against both cutaneous and visceral leishmaniasis. It displayed potent activity with an IC50 value of 18 ± 1.8 nM against intracellular L. donovani amastigotes and an excellent selectivity index compared to mouse peritoneal macrophages (IC50 value >50 µM). Administration of this compound to L. donovani- infected mice via the oral route at a dose of 10 mg/kg twice daily for eight days resulted in 3 times better clearance of liver parasitemia compared to miltefosine administered at a dose of 12 mg/kg once daily. In an L. major induced cutaneous leishmaniasis model, GNF6702 administered at the same dose showed superior results compared to miltefosine administered at a dose of 30 mg/kg once daily. Trials to develop resistant strains of Leishmania species were fruitless, but the authors succeeded in developing resistant strains against Trypanosoma cruzi, another member of the trypanosomatidae family. Whole genome sequencing of the resistant T. cruzi strain showed that there was a mutation in the proteasome. Further studies showed that

GNF6702 selectively inhibited the parasite proteasome with no inhibition of human caspase, trypsin and chymotrypsin proteasome enzymes. Studies with 317 analogs of GNF6702 have shown a positive correlation (r2 value of 0.78) between inhibition of the purified L. donovani proteasome (IC50) and L. donovani axenic amastigote growth inhibition (EC50). Given the

20 promising activity and selectivity of GNF6702 and the importance of the proteasome to eukaryotic cells, GNF6702 might be a good candidate for further preclinical and clinical trials against leishmaniasis [96].

Figure 1.7: Structure of GNF6702

1.6.2 Aminopyrazoles

A series of lead aminopyrazoles were synthesized as antileishmanial drug candidates.

Aminopyrazole urea analogs display in vitro antileishmanial activity in the low micromolar range as well as excellent in vivo activity in the hamster model of visceral leishmaniasis.

Aminopyrazole 1.12 (Figure 1.8) bearing a phenyl piperidine urea group showed IC50 values of

0.296 µM and 0.077 µM against L. infantum and L. donovani in intracellular macrophage assays, respectively. The compound also displayed more than 242 fold selectivity towards the parasite

21 compared to MRC5 human fetal lung fibroblast cells (CC50 value >64 µM). However, aminopyrazole 1.12 displayed poor metabolic stability in the hamster liver microsome assay requiring further modifications. Aminopyrazole 1.13 (Figure 1.8) bearing a fluoro substituted aryl piperazine substituent displayed slightly less potent activity against both intracellular L. infantum and L. donovani amastigotes with IC50 values of 2.37 µM and 1.31 µM, respectively.

Aminopyrazole 1.13 displayed excellent metabolic stability in both human and hamster liver microsomes with intrinsic clearance values of 30.8 and 46.5 µL/min/mg. At an oral dose of 50 mg/kg twice daily for 5 days, treatment with aminopyrazole 1.13 resulted in 92.7% and 95% clearance of liver and spleen parasitemia, respectively, in the hamster model of visceral leishmaniasis. Currently, aminopyrazoles are undergoing further lead optimization studies sponsored by the Drugs for Neglected Diseases initiative (DNDi) to improve physicochemical properties, pharmacokinetics, and solubility [97, 98].

Figure 1.8: Structure of investigated aminopyrazoles

1.6.3 Nitroimidazoles

Nitro compounds have potential toxicity due to the generation of reactive species upon 1 or 2 electron reduction within the body by nitro reductase enzymes [99]. However, the promise of nitro bearing compounds as anti-infectives, such as the anti-tubercular drug delamanid (1.14) and the anti-parasitic fexinidazole (1.15) as shown in Figure 1.9, resulted in the investigation of these nitroaromatic compounds as antileishmanial agents [100, 101]. Fexinidazole sulfone and sulfoxide displayed good in vitro activity against the African visceral leishmaniasis VL9 strain with an IC50 value of 5.3 µM for both fexinidazole metabolites. Oral administration of fexinidazole at 200 mg/kg for 5 days led to 98.4% reduction in liver parasitemia [101].

Fexinidazole was used in phase II clinical trials in combination therapy with miltefosine, however the studies were interrupted due to fertility risk issues with miltefosine [102].

Delamanid showed efficacy comparable to miltefosine in a murine visceral leishmaniasis model with 99.5% reduction in liver parasitemia at an oral dose of 30 mg/kg twice daily for 5 days

[100]. DNDi-VL-2098 (1.16) (Figure 1.9) is another compound with high in vivo potency, but signs of testicular toxicity were observed in animal studies [103]. Currently DNDi-0690, a compound with an undisclosed chemical structure, is in the preclinical phase of antileishmanial development [104].

Fexinidazole and delamanid are cytocidal agents; use of such molecules may be a favorable strategy in treating immunocompromised patients. It is postulated that nitroimidazoles are prodrugs which are activated by nitro reductase enzymes through the production of reactive species damaging cellular components such as DNA, proteins and lipids. In support of this

23 hypothesis, a study showed overexpression of a nitroreductase enzyme in L. donovani amastigotes increased their sensitivity to fexinidazole and delamanid [100, 101].

Figure 1.9: Structures of nitroaromatic antileishmanial candidates

1.7 Introduction to compounds under current investigation

1.7.1 Cyanines

Cyanines (Figure 1.10) are group of zwitterionic compounds consisting of two or more heterocyclic aromatic amino containing compounds with one of them being quaternary. The two ring systems are separated by one or more methine bridges maintaining π electron delocalization

24 through both ring systems [105]. Cyanines have displayed potent antimicrobial activity against a range of protozoan pathogens including the parasites that cause malaria, Chagas disease, African trypanosomiasis and leishmaniasis [106-111]. The rhodacyanine compound MKT-077 (1.17) displayed potent antileishmanial activity in the L. donovani axenic amastigote assay with an IC50 value of 0.255 µM and more than 450-fold selectivity towards the parasite compared to the L-6 rat myoblast cell line (CC50 value of 114.8 µM). Replacement of the 1-ethylpyridinium group with a fluoro substituted benzothiazolium moiety led to 10-23 fold improvement in the antileishmanial activity. SJL-01 (1.18) showed an IC50 value of 0.011 µM in the axenic amastigote assay. SJL-01 displayed submicromolar activity the L. donovani intracellular macrophage assay with an IC50 value of 0.353 µM. SJL-01 showed a selectivity of more than

15,000 fold towards L. donovani axenic amastigotes compared to L-6 rat myoblasts (CC50 value greater than 173 µM). In the L. donovani infected mouse model, no significant antileishmanial activity was observed upon subcutaneous administration of SJL-01, but intraperitoneal administration of 50 mg/kg for 5 days led to 31.43% reduction in liver parasitemia, and intravenous administration of 1.3 mg/kg led to 95.87% reduction in liver parasitemia. Activity was maintained upon removal of the central 4-oxothiazolidine ring as shown in cyanine 1.19.

This compound displayed an IC50 value of 0.020 µM in the in vitro axenic amastigote assay and also retained good selectivity towards the parasite compared to L-6 rat myoblasts (CC50 value of

0.699 µM) [106]. However, these compounds were not subject to any further whole cell in vitro assays or in vivo experiments.

Figure 1.10: Structures of previously tested cyanines

Thiazole orange (1.20) (Figure 1.11) is another cyanine dye that possesses good activity against both L. donovani and L. amazonensis. It consists of benzothiazolium and quinolone ring systems separated by a methine bridge. The antileishmanial activity of thiazole orange was discovered through high throughput screening of approximately 196,000 compounds. Almost 18,000 of these compounds resulted in more than 50% inhibition to L. major promastigotes at concentration of 10 µM which were further categorized in silico into chemical clusters to decrease the actual number of compounds moving to the next stage. A secondary L. major promastigote assay and counter assay against the A549 mammalian epithelial cell line were then conducted, in which 70 compounds displayed an IC50 value less than 1 µM against the

26 promastigotes with no detected toxicity against the A549 cell line at the same concentration

[112]. Further selection yielded 31 compounds by removal of those that showed less than 5 fold selectivity towards L. major promastigotes compared to the A549 cells and exclusion of compounds possessing chemical groups that might lead to toxicity issues such as chloromethyl ketones and Michael acceptors. Leishmania promastigotes provide more affordable preliminary testing of a large set of compounds, yet these parasites do not cause symptoms in the host.

Therefore, these 31 selected compounds were tested against L. donovani and L. amazonensis in intracellular amastigote assays, which evaluate the efficacy of the compounds against the form of the parasite that causes disease in humans. PubChem 123859 (thiazole orange) (1.20) and

PubChem 16196223 (1.21) as shown in Figure 1.11 displayed the best activity in both L. donovani and L. amazonensis assays. PubChem 16196223 is similar to thiazole orange except that the core quinolone ring system was replaced with a pyridinium moiety. Thiazole orange displayed potent activity against intracellular L. donovani with an IC50 value of 0.021 µM, however it displayed intermediate potency against L. amazonensis with an IC50 value of 1.8 µM.

In terms of selectivity, thiazole orange showed more than 66 fold selectivity towards intracellular

L. donovani compared to murine peritoneal macrophages (CC50 value of 1.4 µM). PubChem

16196223 was approximately 10 fold less potent against L. donovani compared to thiazole orange, however it is almost 10 fold more potent against L. amazonensis with IC50 values of 0.18

µM and 0.20 µM respectively. Experiments in L. donovani infected mice showed that both thiazole orange and PubChem 16196223 displayed comparable clearance of liver parasitemia

(44% and 42% respectively at an administered dose of 1 mg/kg intraperitoneally for 5 days), while miltefosine led to 92% and 96% reduction in liver parasitemia at a dose of 10 mg/kg orally

27 or intraperitoneally, respectively, by the same dosing regimen [113]. The methyl β-cyclodextrin vehicle used in the assay displayed antileishmanial activity with 21% inhibition of liver parasitemia, which is not uncommon for β-cyclodextrin. β-cyclodextrin provides a synergistic antifungal activity to amphotericin B by sequestering ergosterol and affecting cell membrane function. Another study showed that methyl β-cyclodextrin depletes the cholesterol content of macrophages decreasing the number of infected cells [114, 115]. A dose response study showed correlation between the dose of thiazole orange administered and the reduction of liver parasitemia. Doses of thiazole orange administered at 0.6 mg/kg or 1.2 mg/kg intraperitoneally for 5 days showed 25% and 48% reduction in liver parasitemia, respectively. Unfortunately, thiazole orange can’t be administered at doses of 2 mg/kg or higher due to toxicity in mice [113].

Figure 1.11: Structure of cyanines discovered by high throughput screening

1.7.2 Arylimidamides

Arylimidamides (AIAs, see Figure 1.12) are a group of chemical compounds that display promising activity against leishmaniasis and Chagas disease. Originally termed reversed amidines, the design of AIAs was based on antimicrobial compounds such as pentamidine containing bisamidino moieties. Furamidine (1.22) is one of these early compounds consisting of bisphenylamidino groups separated by a furyl linker instead of the n-pentyl chain of pentamidine

(1.3). Furamidine shows low nanomolar activity against Plasmodium falciparum and

Trypanosoma b. rhodesiense with IC50 values of 15.5 nM and 4.5 nM, respectively [116].

Further testing of furamidine against L. donovani axenic amastigotes revealed low micromolar potency with an IC50 value of 2.7 µM. Pentamidine shows comparable results with IC50 value of

2.6 µM in the axenic amastigote assay [117]. However, pentamidine showed poor activity in the intracellular L. donovani assay (IC50 value >50 µM). Lack of activity in the intracellular assay is attributed to poor penetration of pentamidine into the parasites. Pentamidine’s amidino groups have pKa values of 11.6. Thus, the compound will be positively charged at physiological pH, making it harder for the compound to cross the macrophage cell membrane as well as the parasitophorous vacuole membranes. By inverting the orientation of the amidino moiety, compounds were prepared where the amino group of the amidine is attached to an aromatic ring, thus lowering its pKa value to approximately 7. This modification led to improvement in the in vitro antileishmanial activity against intracellular Leishmania, resulting in sub micromolar IC50 values [118]. Of the first set of AIAs, the imidamide 1.23 containing bis(3-methylthiophenyl) furan as a linker and bis-pyridyl peripheral aromatic ring showed the best antileishmanial activity with IC50 values of 0.35 µM and 0.15 µM in the L. donovani axenic amastigote and intracellular

29 assays, respectively [117]. Further modifications resulted in the frontrunner AIA DB766 (N,N''-

(furan-2,5-diylbis(3(2-propoxy)-4,1-phenylene))dipicolinimidamide) (1.24), which displayed

IC50 values of 0.5 µM and 0.036 µM against L. donovani axenic amastigotes and the L. donovani

LV82 strain in the intracellular assay, respectively. DB766 also displayed potent activity against intracellular L. amazonensis (IC50 value of 0.087 µM) [118].

Figure 1.12: Structures of pentamidine and previously synthesized imidamide analogs

Various modifications were performed both on the bisphenyl furyl linker as well as the terminal aromatic ring as shown in Figure 1.13. Regarding the linker, smaller arylimidamide analogs were synthesized by removal of the core furyl ring (analog 1.25) or by replacing it with oxygen

(analog 1.26) leading to good in vitro antileishmanial activity. Unfortunately, these compounds proved to be very toxic to mice. Replacement of the furyl ring with a methylene group (analog

1.27) led to loss of in vitro antileishmanial activity [119]. Isosteric replacement of the furan ring with thiophene in DB1867 (1.28) led to comparable antileishmanial activity with IC50 values of

0.68 µM and 0.045 µM against L. donovani axenic amastigotes and intracellular L. amazonensis, respectively [120]. Isosteric replacement of furan with a phenyl group gave the meta-terphenyl analog 1.29 which was tested only against L. amazonensis [121]. Analog 1.29 showed good antileishmanial activity, albeit weaker than that of DB766. Substitutions on the furan ring were also investigated; these compounds differ from DB766 by lacking the bis(isopropoxy) groups as well as introducing a hydrophobic group represented by phenyl or methyl groups on the furyl ring. 3-Phenyl substituted furyl analog DB889 (1.30) was less potent than DB766 while 3,4- dimethyl substituted furyl analog DB946 (1.31) showed comparable in vitro activity to DB766 against L. donovani axenic amastigotes and intracellular L. amzonensis [120]. Substitutions on the bisphenyl moieties with alkyl, halogen and other alkoxy groups were performed to give a thorough understanding of arylimidamide structure activity relationships. Replacement of isopropoxy with methoxy or ethoxy groups yielded analogs DB709 (1.32) and DB745 (1.33) respectively which displayed potent antileishmanial activity against both L. donovani and L. amazonensis in vitro [118, 120]. However, DB745 displayed greater toxicity in mice compared to DB766 [118]. The use of bulkier substituents instead of isopropoxy such as isobutoxy, tert- butoxy, cyclopentyloxy and benzyloxy led to a decrease in antileishmanial activity against L. donovani axenic amastigotes and L. amazonensis intracellular amastigotes, especially for the benzyloxy group (more than a 10 fold decrease in activity) [120]. Unsymmetrical

31 arylimidamides, where one of the isopropoxy groups was replaced with halogen, methyl or methoxy groups, were active, however these compounds were toxic to mice when given at an oral dose of 30 mg/kg [122].

Further modifications were performed on the terminal pyridyl groups. Introduction of an electron donating methoxy or methyl group at the 2 or 3 position of the pyridyl ring led to a drop in antileishmanial activity against L. donovani and L. amazonensis. This decrease in potency was particularly striking for the compound DB1907 (1.34) bearing the ortho-methoxy group which displayed IC50 values of 4.5 µM and 2.7 µM against L. donovani axenic amastigotes and L. amazonensis intracellular amastigotes. The 3-bromo substituted pyridyl analog DB1889 (1.35) was also less potent, with an IC50 value >10 µM against L. donovani and L. amazonensis.

Replacement of the pyridyl ring by other heteroaromatic groups such as pyrimidyl, pyrazinyl, pyrrolyl, imidazolyl, and substituted thiazolyl rings led to a reduction in antileishmanial activity.

Incorporation of a 2-methylthiazolyl group in analog DB1850 (1.36) instead of the 2-pyridyl ring led to only a twofold drop in activity against L. amazonensis intracellular amastigotes with an IC50 value of 0.17 µM. Replacement of the pyridyl ring by a methyl group led to diminished antileishmanial activity against both L. donovani and L. amazonensis [120, 123]. Analogs with a single arylimidamide group (mono arylimidamides) were also investigated. DB2002 (1.37), which contains one less pyridylimidamide and isopropoxy group compared to DB766 showed potent antileishmanial activity with an IC50 value of 0.31 µM and 0.13 µM against L. donovani and L. amazonensis intracellular amastigotes, respectively. Oral administration of 50 mg/kg and

100 mg/kg of DB2002 for 5 days resulted in 37% and 46% inhibition of liver parasitemia in the

32 murine visceral leishmaniasis model. The submicromolar in vitro activity of the mono arylimidamides allows a new avenue for future structural modification of arylimidamides [124].

Figure 1.13: Modifications performed on the lead compound DB766

As previously mentioned, DB766 displayed one of the best antileishmanial activities of the AIAs against both L. donovani and L. amazonensis with an IC50 value in the nanomolar range and also possesses potent activity against antimony resistant L. donovani strains. DB766 displayed good selectivity towards the parasite compared to murine peritoneal macrophages (CC50 value of 2.8

µM). The activity of DB766 might be attributed to its physicochemical properties; it is more lipophilic and its amidino group is less basic compared to pentamidine. DB766 has a distribution coefficient (Log D7.4) value of 4.12 and pKa close to physiological pH with a value of 7.36.

DB766 was tested in both hamster and mouse models of visceral leishmaniasis. Oral administration of DB766 at 100 mg/kg in L. donovani infected mice for five consecutive days resulted in 71% reduction of liver parasitemia. DB766 displayed dose-dependent activity as a 50 mg/kg oral dose for the same period of time led to 41% reduction in liver parasitemia. DB766 showed minor hepatic toxicity at the administered dose of 100 mg/kg in the mouse model, yet all treated animals survived [118]. The hamster model more closely resembles visceral leishmaniasis in humans compared to the mouse model, yet it is more expensive and typically makes use of intracardial infection, making the mouse model the first choice for assessing in vivo antileishmanial activity. Oral administration of DB766 at a dose of 100 mg/kg for 5 consecutive days resulted in 89%, 79% and 92% reduction in liver, spleen and bone marrow parasitemia in the hamster model, respectively [118]. Since DB766 had limited aqueous solubility, allowing only the testing of doses up to 100 mg/kg, DB1960 (the mesylate salt of

DB766) was administered at a higher dose. Oral administration of DB1960 at 50 mg/kg, 100 mg/kg and 200 mg/kg for five consecutive days in L. donovani infected mice led to 29%, 51% and 93% reduction in liver parasitemia. However, animals receiving 100 mg/kg oral doses of

DB1960 displayed histopathological changes in all organs examined, while changes in serum chemistry values were also observed in mice receiving 200 mg/kg and 500 mg/kg doses of this compound, indicating that DB766 provides better therapeutic index [125]. Unfortunately, the failure to clear parasitemia in these models indicated that further optimization is needed.

1.7.2.1 Investigation of the antileishmanial mechanism of arylimidamide DB766.

The mechanism of action of pentamidine is not fully understood; however, studies have reported that it targets kinetoplastid DNA as a minor groove binder [126] and also interferes with the parasite mitochondrion [127]. Pentamidine and other diamidines such as furamidine have shown

DNA binding activity which was confirmed by X-ray crystallography using synthetic poly AT rich DNA. Also, pentamidine and furamidine cause a significant change in the DNA melting temperature, providing further support for DNA binding as one mechanism of diamidine action.

However, studies with arylimidamides didn’t show any correlation between the in vitro antileishmanial activity and the change in DNA melting temperature [117, 118]. DB766 caused a minor increase in the melting temperature of synthetic poly AT DNA (6 ºC) [118]. Pandharkar et al. demonstrated through electron microscopy studies that the arylimidamide DB766 has different cellular targets than the diamidine DB1111 due to the distinct effects on Leishmania cellular morphology caused by the two agents [128]. Also, in a DB766 resistant strain of L. donovani raised by DB766 pressure in culture, cross resistance to pentamidine was not observed.

Interestingly, this DB766 resistant L. donovani strain was hypersensitive to the azoles posaconazole and ketoconazole. Further studies showed that decreased expression of the

CYP5122A1 enzyme with a slight increase in CYP51 expression (14-α demethylase) was

35 observed in the DB766 resistant strain [128]. Although the precise role of CYP5122A1 in

Leishmania is unknown, it is an essential enzyme in the parasite. Knockout of both CYP5122A1 alleles was lethal in L. donovani, while half knockout parasites contained a 3 fold decrease in ergosterol levels [129]. Half knockout L. donovani were more resistant to the effects of DB766 and more sensitive to ketoconazole than the corresponding wild type parasites, similar to the

DB766 resistant L. donovani strain. Thus, although more work is needed to further clarify the mechanism of action of DB766, this compound may exert its antileishmanial activity by interfering with CYP5122A1 activity or may be metabolized to a more active species by this novel protein [128].

Chapter 2

Design and synthesis of cyanines as antileishmanial agents

2.1 Aim of the work

As mentioned earlier, thiazole orange displayed potent in vitro activity in the L. donovani intracellular assay, yet it showed toxicity against murine peritoneal macrophages in the low micromolar range and was toxic to mice at an i.p. dose of 2 mg/kg/day [113]. Our plan is based on the phenotypic screening of synthesized analogs of thiazole orange aiming to improve selectivity towards the parasite. The synthetic medicinal chemistry approach is based on the

Topliss tree [130, 131] which is a simple method for analog design developed by Prof. J. G.

Topliss. It is an empirical method to develop the structure activity relationship of group of synthesized analogs instead of performing computational calculations or the synthesis of an extensive group of analogs. The Topliss tree is based on the Hansch equation and it addresses the electronic, lipophilic and steric properties of substituents on a phenyl ring or a benzenoid moiety of a fused heterocyclic ring. Substituent choice is based on feasibility of the synthetic procedures as well as the physicochemical properties of the proposed analogs.

An initial set of 12 compounds 2.1e-p (Scheme 2.6) were synthesized bearing chloro, methyl or methoxy groups on the benzenoid moiety of the quinoline ring system of thiazole orange at positions 5-8 following the Topliss tree batch approach. Three analogs bearing one less phenyl ring 2.1u-w (Scheme 2.7 and Scheme 2.8) were synthesized since one of the cyanine hits from our screening, PubChem 16196223 (1.21) (Figure 1.11) bearing a pyridyl moiety instead of quinolinyl group, displayed sub-micromolar potency against intracellular L. donovani and L. amazonensis [113]. Four other analogs 2.1q-t (Scheme 2.6) were synthesized by placing a phenyl ring on the benzothiazolium moiety to examine the effect of an additional aromatic ring on biological activity. Analogs were also prepared bearing substitutions on the quinoline nitrogen atom 2.1b-d (Scheme 2.6) since the commercial SYBR Safe dye (2.1c with a tosylate counter ion instead of the iodide) bearing an n-propyl group at this position was reported to be less toxic than thiazole orange [132].

Figure 2.1 Proposed cyanine analogs

2.2 Chemistry

Scheme 2.1 highlights the synthesis of 1,4-dimethyl quinolines bearing electron donating groups following Knorr’s quinolone synthesis [133]. Briefly, o-, m- and p-substituted anilines reacted with ethyl acetoacetate under neat conditions followed by acid catalyzed cyclization using sulfuric acid or the milder polyphosphoric acid to yield compounds 2.4a-g in low yields [134-

136]. Wlodarczyk et al. [137] mentioned that the synthesis of anilides by Knorr’s method was achieved in low yield due to side products. Such impurities were separated by column chromatography or simply by trituration from ether [134]. Compounds 2.5a-g were synthesized

39 in excellent yields firstly by halogenation using phosphorous oxychloride [138, 139] followed by transfer hydrogenation employing palladium on carbon using hydrazine as the hydrogen source

[139]. Finally the dimethyl quinolinium salts 2.6a-j were synthesized by reaction with different alkyl iodides under sealed tube conditions.

Scheme 2.1: Synthesis of 2.6a-j. Reagents and conditions: (a) neat, 180 °C, 24-48 h; (b) PPA, 95

1 °C, 3h; (c) POCl3, reflux, 2-3 h; (d) NH2NH2·H2O 80%, Pd/C 10%, EtOH, reflux, 4-8 h; (e) R I,

MeOH, sealed tube, 80 °C, 2-8 h.

While the 6, 7 and 8-substituted lepidines (4-methylquinolines) could be made by either the

Knorr quinolone synthesis or Skraup-Doebner-Von Miller quinoline synthesis, neither of them could be used directly for the synthesis of the 5-substituted quinolines as the m-substituted anilines will predominantly lead to the 7-substituted lepidine rather than the 5-substituted isomer.

Thus, a blocking strategy was employed as outlined in Scheme 2.2 starting with the reduction of the commercially available ortho substituted chloronitrobenzenes 2.7a-b to give the corresponding anilines 2.8a and 2.8b [140]. The Skraup-Doebner-Von Miller quinoline synthesis was then utilized for the preparation of 5-substituted-8-chloro lepidines followed by dehalogenation to yield 2.5h and 2.5i. The quinolinium salts 2.6k and 2.6l were synthesized by the reaction of 2.5h and 2.5i with methyl iodide.

Scheme 2.2: Synthesis of 2.6k,l. Reagents and conditions: (a) Fe, acetic acid, rt to 90 °C, 30 min;

(b) methyl vinyl ketone (2.9), dioxane, H2SO4, reflux, 24 h; (c) NH2NH2·H2O 80%, Pd/C 10%,

EtOH, reflux, 4-8 h; (d) CH3I, MeOH, sealed tube, 80 °C, 2-4 h.

Scheme 2.3 shows the Skraup-Doebner-Von Miller quinoline synthesis [141] used for lepidines bearing electron withdrawing substituents. Anilines were reacted with methyl vinyl ketone under acidic conditions to yield compounds 2.11a-f [142]. Formation of the corresponding 1,4 dimethyl quinolinium salts 2.12a and 2.12b was achieved through N-methylation using methyl

41 iodide. Since the reaction of compound 2.11c with methyl iodide was sluggish, methyl triflate was employed for the synthesis of the quinolinium salt 2.12c [143]. Methyl triflate was used with great caution considering its strength as an alkylating agent.

Scheme 2.3: Synthesis of 2.12a-c. Reagents and conditions: (a) dioxane, H2SO4, reflux, 48 h; (b)

CH3I, MeOH, sealed tube, 80 °C, 4-8 h; (c) CF3SO3CH3, rt, 24 h.

Synthesis of 5-halo substituted lepidines (2.11g,h) couldn’t be performed directly by Knorr’s method and another synthetic pathway was considered starting from 2.5i. Although demethylation of 2.5i [144] yielded the corresponding 5-hydroxylepidine, chlorination of this derivative to achieve the 5-chlorolepidine was unsuccessful and an alternative route was required as shown in Scheme 2.4. Nitration of 8-bromolepidine (2.11f) employing potassium nitrate and sulfuric acid gave compound 2.13 followed by reduction and dehalogenation to prepare the 5-

42 aminolepidine (2.14) whose structure was confirmed by 2D COSY and NOE NMR experiments.

The 5-halo derivatives 2.11g and 2.11h were synthesized by Sandmeyer reaction using copper(I) chloride and copper(I) bromide respectively [145, 146]. Compound 2.12d was prepared in excellent yield by reaction of 2.11g with methyl iodide.

Scheme 2.4: Synthesis of 2.12d. Reagents and conditions: (a) H2SO4, KNO3, 0 °C to rt, 20 h; (b)

Fe, acetic acid, rt to 90 °C, 30 min; (c) NH2NH2·H2O 80%, Pd/C 10%, EtOH, reflux, 8 h; (d)

NaNO2, HCl, CuCl, -10 °C to rt, 10 h; (e) NaNO2, 48% HBr, CuBr, -10 °C to rt, 10 h; (f) CH3I,

MeOH, sealed tube, 80 °C, 2-4 h.

Scheme 2.5 outlines the synthesis of the quinolinium salts bearing a phenyl group at positions 5-

8 (2.15a-d) via Suzuki coupling [147] between phenyl boronic acid and the bromo analogs

2.11d-f and 2.11h, followed by N-methylation as discussed previously.

Scheme 2.5: Synthesis of 2.16a-d. Reagents and conditions:(a) PhB(OH)2, Pd[P(Ph)3]4, EtOH,

Cs2CO3, reflux, 12 h; (b) CH3I, MeOH, sealed tube, 80 °C, 2-4 h.

Scheme 2.6 displays the synthesis of cyanines following previously published procedures with slight modifications [148, 149]. Methylation of the commercially available 2-methylthio benzothiazole (2.17) with methyl iodide or methyl triflate provided 2.18a and 2.18b.

Triethylamine-catalyzed condensation of 2.18a and 2.18b with the quinolinium salts 2.6a-l, 2.9a- d and 2.16a-d yielded the corresponding cyanine dyes 2.1a-t.

Scheme 2.6: Synthesis of 2.1a-t. Reagents and conditions: (a) CH3I, MeOH, sealed tube, 80 °C,

8 h; (b) CF3SO3CH3, rt, 24 h; (c) NEt3, EtOH: DMF (1:1), reflux, 12 h.

The synthesis of thiazolyl cyanine 2.1u (Scheme 2.7) and pyridyl cyanines 2.1v and 2.1w

(Scheme 2.8) followed previously published procedures [150, 151]. 2-(Methylthio)thiazole

(2.20) was converted to the corresponding thiazolium salt (2.21), then stirring 2.21 with 1,4- dimethylquinolinium iodide 2.6a in dichloromethane at room temperature with 5 equivalents of triethylamine yielded compound 2.1u (Scheme 2.7). In similar fashion, 2.23a and 2.23b

[obtained by methylation of 2-picoline (2.22a) or 4-picoline (2.22b)] reacted with 2.18a to obtain the cyanines 2.1v and 2.1w, respectively (Scheme 2.8).

Scheme 2.7: Synthesis of 2.1u. Reagents and conditions: (a) CH3I, MeOH, sealed tube, 80 °C, 8 h; (b) 2.6a, NEt3, DCM, rt, 48 h.

Scheme 2.8: Synthesis of 2.1v,w. Reagents and conditions: (a) CH3I, MeOH, sealed tube, 80 °C,

2-4 h; (b) 2.18a, NEt3, DCM, rt, 48 h.

2.3 Crystal structure of 2.1q

To examine the geometry of the cyanine target compounds, an X-ray crystal structure was obtained for 2.1q (Figure 2.3) which was performed by Dr. Slebodnick at Virginia Tech. This crystal structure shows the quinoline and benzothiazole ring systems to be essentially coplanar, as expected from the delocalized π-electron system present in the compound and in agreement with a previous report [152]. The conformation of the quinoline and thiazole ring systems are similar to the conformation observed based on Nuclear Overhauser data and the calculated low energy conformation for thiazole orange reported earlier [132]. In the crystal structure, the phenyl ring at position 5 of 2.1q is roughly orthogonal to the plane of the cyanine ring system.

Figure 2.2: X-ray crystal structure of 2.1q.

2.4 Biological data

IC50 and CC50 determinations were made by Xiaoping Liao, Kelsey Bryant and Dr. April Joice.

To provide a clear picture of the inherent selectivity of the cyanine target compounds for

Leishmania compared to a mammalian cell line, the effect of these compounds on L. donovani axenic amastigotes and J774 murine macrophages was measured (Table 1). Cyanine target compounds displayed exceptional potency against L. donovani axenic parasites, with IC50 values ranging from 0.012 µM to 0.29 µM. Among compounds substituted on the quinoline ring system of the cyanine scaffold, structural modifications had little effect on antileishmanial potency. For example, the placement of different substituents at the quinoline nitrogen atom ranging in size from a methyl group to a benzyl group (2.1a-d) resulted in antileishmanial potencies varying only by 2.5-fold. Similarly, within the series of compounds substituted at positions 5-8 on the quinoline ring system with either a methyl (2.1e-h), methoxy (2.1i-l), chlorine (2.1m-p), or phenyl group (2.1q-t), antileishmanial potencies varied by only 1.5-, 1.5-,

2.1-, and 4.2-fold, respectively. While in general the substitution on the quinoline ring system had little effect on antiparasitic activity, substitution with phenyl rings at positions 5 and 6 of the quinoline core caused a slight decrease in antiparasitic activity. Despite retaining significant activity against L. donovani, substitution of a thiazole ring (2.1u) for the benzothiazole system led to an 11-fold decrease in activity against L. donovani axenic amastigotes compared to parent compound 2.1a. Likewise, compounds where a 4-pyridyl substituent (2.1v) or a 2-pyridyl substituent (2.1w) replaced the quinoline are less potent than the corresponding 2.1a by 8.6- to

20-fold. Cyanine derivatives 2.1a-w were in all cases less toxic to J774 macrophages than to L. donovani axenic amastigotes, although they nonetheless displayed low CC50 values against this

48 mammalian cell line (CC50 values ranging from 0.053 µM to 2.8 µM). Selectivity indexes ranged from 1.8 to 20 for synthesized analogs. Analogs 2.1b, 2.1v and 2.1w with the best selectivity indexes towards the axenic parasite were tested against intracellular L. donovani amastigote were they showed approximately 5 fold decrease in potency in the whole cell assay compared to the axenic amastiogtes assay.

Table 2.1: In Vitro Potency of Target Cyanines 2.1a-p against L. donovani Axenic Amastigotes and J774 Macrophages

b Comp. R R1 Axenic Ld Intracellular Ld J774 SI amastigotes amastigotes a IC50 in µM TO (2.1a) Me H 0.014 ± 0.000 - 0.19 ± 0.03 14 2.1b Et H 0.012 ± 0.002 0.072 ± 0.009 0.17 ± 0.03 14 2.1c Pr H 0.013 ± 0.001 - 0.14 ± 0.02 11 2.1d Bn H 0.026 ± 0.004 - 0.19 ± 0.03 7.3 2.1e Me 5- Me 0.024 ± 0.002 - 0.12 ± 0.01 5.0 2.1f Me 6- Me 0.031 ± 0.003 - 0.20 ± 0.03 6.5 2.1g Me 7- Me 0.037 ± 0.003 - 0.12 ± 0.02 3.2 2.1h Me 8- Me 0.030 ± 0.006 - 0.12 ± 0.02 4.0 2.1i Me 5-OMe 0.025 ± 0.003 - 0.086 ± 0.012 3.4 2.1j Me 6-OMe 0.028 ± 0.002 - 0.20 ± 0.02 7.1 2.1k Me 7-OMe 0.035 ± 0.002 - 0.14 ± 0.01 4.0 2.1l Me 8-OMe 0.030 ± 0.002 - 0.053 ± 0.010 1.8 2.1m Me 5-Cl 0.016 ± 0.003 - 0.13 ± 0.02 8.1 2.1n Me 6-Cl 0.031 ± 0.002 - 0.31 ± 0.04 10 2.1o Me 7-Cl 0.034 ± 0.003 - 0.18 ± 0.02 5.3 2.1p Me 8-Cl 0.020 ± 0.005 - 0.11 ± 0.01 5.5 Pentamidine - - 3.1 ± 0.2 - - - Podophyllotoxin - - - - 0.023 ± 0.002 - Amphotericin B - - - 0.036 ± 0.002 - - aMean ± standard error of at least three independent determinations b CC50 J774/IC50 axenic Ld

Table 2.2: In Vitro Potency of Target Cyanines 2.1q-w against L. donovani Axenic Amastigotes and J774 Macrophages

Comp. R R1 Axenic Ld Intracellular Ld J774 SI amastigotes amastigotes a IC50 in µM 2.1q Me 5-Ph 0.042 ± 0.010 - 0.16 ± 0.03 3.8 2.1r Me 6-Ph 0.037 ± 0.011 - 0.18 ± 0.03 4.9 2.1s Me 7-Ph 0.013 ± 0.003 - 0.069 ± 0.009 5.3 2.1t Me 8-Ph 0.024 ± 0.003 - 0.20 ± 0.04 8.3 2.1u - - 0.16 ± 0.01 - 1.5 ± 0.1 9.4 2.1v - - 0.14 ± 0.03 0.79 ± 0.07b 2.8 ± 0.2 20 2.1w - - 0.29 ± 0.02 1.3 ± 0.2b 2.5 ± 0.3 8.6 Pentamidine - - 3.1 ± 0.2 - - - Podophyllotoxin - - - - 0.023 ± 0.002 - Amphotericin B - - - 0.036 ± 0.002 - - aMean ± standard error of at least three independent determinations bMean ± range of two independent determinations

Our analysis of the 22 synthesized cyanines related to 2.1a confirmed the exceptional in vitro antileishmanial potency of the class. However, the SAR in this series is relatively flat when substitutions on the quinolinium ring are examined. Removal of one aromatic ring from either the quinolinium or benzothiazole portion of the scaffold led to a decrease in toxicity but also a decrease in potency. While selectivity was observed for the cyanines for L. donovani compared to the J774 cell line, antileishmanial activity generally correlated with cytotoxicity (Table 2.1 and Table 2.2). Thus, the cyanines possess some of the characteristics of nonspecific drugs [153]

51 where a specific site of action is lacking and potency is less dependent on small structural changes. Thus, despite the high potency of these benzothiazole cyanines, our current investigation was not successful in dissociating antiparasitic potency from toxicity.

2.5 DNA binding studies

DNA binding studies was performed by Dr Guo in Prof. David Wilson lab at Georgia State

University. The ability of selected cyanine target compounds to bind double-stranded DNA was assessed by measuring the increase in melting temperature of two mixed sequence synthetic hairpin DNA oligomers (5'-dCCAAAATTTTG CTCT CAAAATTTTGG-3' [AAAATTTT]; 5'- dCCAAAAGTTTTG CTCT CAAAACTTTTGG [AAAAGTTTT] with the hairpin CTCT loop underlined) that have been used in previous studies with synthetic compounds [154]. Circular dichroism studies were also conducted with a representative cyanine, 2.1f, binding to an AT rich

DNA. A negative induced CD signal was observed, which is characteristic of an intercalation binding mode [155]. For Tm determinations the synthetic compounds were incubated with the

DNA samples prior to Tm measurements as previously described [154, 156]. The compounds chosen for this analysis examined the effect on DNA binding of placing a methyl group at positions 5-8 of the quinoline ring (2.1e-h) in addition to placing the phenyl group at position 5

(2.1q). The truncated derivatives 2.1v, 2.1w, and 2.1u were also tested in this assay. For the oligomer containing the double-stranded AAAATTTT sequence, compounds increased the DNA

Tm by only 3-5 degrees. Truncated derivatives 18u-v were poorer binders to this oligomer, increasing Tm by 2 degrees or less. The other DNA gave similar, relatively low, elevations in Tm with melting temperature increases below 5 °C in the presence of these compounds. The

52 truncated derivatives gave the lowest increase in melting temperature of all of the derivatives.

The relatively low Tm values of these DNA complexes suggests that DNA binding is not the major driver their antileishmanial potency, although DNA binding may be one component in a complex mechanism of action of these compounds.

Figure 2.3: Circular dichroism titration spectra of 2.1f

Table 2.3: DNA binding of selected cyanines to synthetic double stranded oligomers

ΔTm (°C) for AAAATTTT 2.1a 2.1e 2.1f 2.1g 2.1h 2.1q 2.1u 2.1v 2.1w 4 3 5 4 4 3 2 1 <1 ΔTm (°C) for AAAAGTTTT 2.1a 2.1e 2.1f 2.1g 2.1h 2.1q 2.1u 2.1v 2.1w 4 4 4 4 3 2 1 <1 <1

2.6 Cyanines mechanism of action

It has been proposed that rhodacyanines act by disrupting the mitochondrial membrane potential in the parasite [108]. This proposal is based on the action of rhodacyanine anticancer compounds such as MKT-077, which was shown to accumulate in the mitochondrion of cancer cells to a greater extent than in normal cells [157] in accord with the delocalized lipophilic cation hypothesis outlined by Chen [158]. Studies employing fluorescent rhodacyanine antimalarial compounds showed the selective and irreversible accumulation of these molecules within P. falciparum infected erythrocytes compared to non-infected erythrocytes. The most potent rhodacyanine compounds accumulated in specific organelles while less potent derivatives exhibited diffuse staining throughout the parasite cytoplasm; double staining experiments revealed that a potent antimalarial rhodacyanine colocalized with MitoTracker Red CMXRos but not with the nuclear diamidine stain DAPI [107]. Although the antileishmanial mechanism of action of these compounds has not been investigated, a similar mitochondrial-directed mechanism seems likely. Since we could not dissociate antiparasitic potency from toxicity and since a low dose of 2.1a was not well tolerated in mice [113], we concluded that further investigation with this class of cyanines is unlikely to bear fruit in the discovery of new antileishmanial compounds in the absence of further information allowing the more specific targeting of these agents to parasites.

Chapter 3

Design and synthesis of arylimidamide azole hybrids as antileishmanial agents

3.1 Aim of the work

Since DB766 (1.24) displayed the best in vitro and in vivo antileishmanial activity of synthesized arylimidamide analogs, and L. donovani parasites resistant to DB766 were hypersensitive to orally active antifungal drugs posaconazole and ketoconazole, combinations of these agents were evaluated for activity against L. donovani in vitro. Since both azoles enhanced the activity of

DB766, it was used in combination therapy studies with both posaconazole and ketoconazole. In the DB766-posaconazole study, 16 groups containing four mice per group were employed.

DB766 and posaconazole were given at 3 different doses (75, 37.5 and 18.8 mg/kg for DB766 and 30, 15 and 7.5 mg/kg for posaconazole). Six groups received monotherapy of either DB766 or posaconazole, one group received PEG 400 as a control group, and 9 groups received combinations of both compounds. DB766 and posaconazole were given at three different doses as indicated in Figure 3.1. A synergistic effect was observed for four groups, two groups displayed a moderately synergistic effect, while the last three groups displayed an additive effect as assessed by the combination index method [159]. In vivo synergism was observed between

55 posaconazole and DB766, while in a separate experient with a combination of DB766 and ketoconazole examined in the murine model of visceral leishmaniasis, mixed results ranging from synergism to antagonism were observed for the DB766-ketoconazole groups [160].

Pharmacokinetic studies showed that posaconazole as a CYP inhibitor increases the plasma concentration of DB766. This subsequently led to the increased concentration of DB766 in livers of infected mice. It was found that the combination 37.5 mg/kg/day DB766 and 15 mg/kg/day posaconazole led to three and two fold increase in the liver concentration of DB766 and posaconazole respectively compared to monotherapy of either drug alone. A pharmacokinetic study of the DB766-ketoconazole combination showed that DB766 blocked absorption of ketoconazole, providing a possible explanation for the different result observed with this combination in the mouse model of visceral leishmaniasis. A combination treatment approach with DB766 and posaconazole might be attractive due to inhibition of DB766 metabolism and improvement of its pharmacokinetic properties [160].

Figure 3.1: In vivo efficacy of DB766-posaconazole combinations [reprinted with the permission

from American Society for Microbiology: Antimicrob. Agents Chemother.][160]

In the current research project, we would like to approach co-administration of an arylimidamide with an azole from another perspective through a hybridization approach [161]. Inclusion of both an azole and imidamide moiety in a single compound might have a positive impact on the in vivo pharmacokinetics and/or pharmacodynamics of these compounds, and such hybrid molecules might interact with the targets of both classes of compounds. Hybridization has previously been employed for the development of antileishmanial agents (Figure 3.2). Hybrid analogs incorporating the pentamidine scaffold and features of aplysinopsin (3.1) displayed good activity against L. donovani in vitro. Aplysinopsin is a cyclic natural product isolated from the Jamaican

57 sponge Smenospongia aurea; it possesses good activity against P. falciparum and

Mycobacterium tuberculosis [162]. The hybrid analog 3.2 shown in Figure 3.2 possesses the amidoxime modification of pentamidine (1.3) at one end and the E-configuration of aplysinopsin at the other. This compound displayed an IC50 value of 2.0 µM against intracellular L. donovani amastigotes with over 50 fold selectivity with respect to the KB cell line (CC50 value of 108.5

µM). In vivo evaluation of hybrid 3.2 led to 62.1 ± 9.8% reduction in liver parasitemia in a hamster model of visceral leishmaniasis [163]. Hybrid analogs possessing carboline and tetrazole moieties have displayed low micromolar activity against both L. donovani promastigotes and intracellular amastigotes. There are a number of natural product based compounds containing the

β-carboline scaffold with good antileishmanial activity, such as annomontine (3.3), and harmine

(3.4) as well as tetrazole PD 12 (3.5) which possess a tetrazole moiety. Hybrid analog 3.6 with tetrahydro-β-carboline and tetrazole pharmacophores displayed an IC50 value of 1.57 µM against

L. donovani intracellular amastigotes and more than 20 fold selectivity towards J774 macrophages compared to the parasite (CC50 value 36 µM) [164].

Figure 3.2 Examples of hybrid analogs with antileishmanial activity, along with some

compounds that inspired their synthesis.

Thus based on the promising in vivo antileishmanial efficacy of the posaconazole/DB766 combination and the good in vitro activity of mono arylimidamides, we proposed the synthesis of hybrid molecules containing the pyridylimidamide and azole groups (Figure 3.3). A preliminary group of 10 compounds (3.12a-j) (Scheme 3.1) bearing the imidazole or triazole moiety together

59 with the pyridylimidamide group were synthesized. The azole and imidamide were separated by a phenoxy alkyl linker with a chain length ranging from ethyl to decyl to determine the effect of homologation on biological activity. The most active compound in the series was subjected to modification by replacing the pyridyl ring of the imidamide scaffold with phenyl ring 3.12k

(Scheme 3.2) or the azole moiety with a pyrrole ring 3.12l (Scheme 3.2) to shed further light on the structure activity relationship. Substitution at the ortho or meta positions of the phenoxy ring system is also made with either a chloro 3.12m,n (Scheme 3.3) or small alkoxy group 3.20a-c

(Scheme 3.4) and 3.27a-c (Scheme 3.5). Since structural rigidity can provide better drug like properties, biphenyl alkyloxy linkers with shorter alkyl chains (butyl or hexyl, as the extra phenyl ring is comparable in length to a three carbon chain) have also been incorporated in hybrid molecules 3.33a,b (Scheme 3.6). Substitution has also been made at the 2, 2', 3 or 3' positions of the biphenyl moiety with methoxy or isopropoxy groups 3.33c-j (Scheme 3.6),

3.42a-d (Scheme 3.7), and 3.51a-d (Scheme 3.8). An additional two analogs 3.51e,f (Scheme

3.8) possessing methoxy or isopropoxy groups at both the 3 and 3' positions were prepared as this substitution might disrupt the planar configuration of the biphenyl group.

Figure 3.3 Hybrid synthetic strategy

3.2 Chemistry

Scheme 3.1 shows the synthesis of AIA azole hybrids 3.12a-j. According to a previously published procedure [165], nucleophilic substitution of dibromo alkanes of different chain lengths with 3.8a in dry acetonitrile under basic conditions yields 3.9a-g followed by nucleoophilic attack by imidazole or triazole to give compounds 3.10a-j under the same basic conditions as for the preparation of 3.9a-g. Nitro reduction employing tin chloride yielded 3.11a- j which were found to be unstable. These arylamines were thus taken directly to the next step without any further purification, reacting with the thioimidate salt under conditions published previously to yield target compounds 3.12a-j.

Scheme 3.1: Synthesis of 3.12a-j. Reagents and conditions: a) Dibromoalkane, K2CO3, CH3CN, reflux; b) imidazole or 1,2,4-triazole, K2CO3, CH3CN; c) SnCl2·2H2O, EtOAc; d) S-(2- naphthylmethyl)-2-pyridylthioimidate·HBr, CH3CN/EtOH (1:3), rt.

Scheme 3.2 shows the synthesis of AIA azole hybrids possessing different terminal moieties.

Compound 3.12k possesses a phenyl group instead of a pyridyl group at the imidamide end while compound 3.12l contains a pyrrole group instead of triazole or imidazole at the azole terminus. Synthesis of these molecules is similar to the route shown in Scheme 3.1 except that the phenyl thioimidate salt is used instead of the pyridyl thioimidate salt to obtain target compound 3.12k. Also, the nucleophilic attack of pyrrole on 3.9g required the stronger base

KOH to abstract the weakly acidic NH proton of pyrrole. The polar solvent DMF is also used to facilitate the reaction [166].

Scheme 3.2: Synthesis of 3.12l,m. Reagents and conditions: a) 1,8-dibromooctane, K2CO3,

CH3CN, reflux; b) imidazole, K2CO3, CH3CN, reflux; c) Pyrrole, KOH, DMF, reflux d)

SnCl2·2H2O, EtOAc; e) Naphthalen-2-ylmethyl benzimidothioate·HBr, CH3CN/EtOH (1:3), rt.; f) S-(2-naphthylmethyl)-2-pyridylthioimidate·HBr, CH3CN/EtOH (1:3), rt.

Scheme 3.3 shows the synthesis of AIA azole hybrids 3.12m,n with a chlorine atom substituted either at the ortho or meta positions to the imidamide group. Synthesis starts with 2-chloro- or 3- chloro-4-nitrophenol following the same synthetic procedure as in Scheme 3.1.

Scheme 3.3: Synthesis of 3.12m,n. Reagents and conditions: a) 1,8-dibromooctane, K2CO3,

CH3CN, reflux; b) imidazole, K2CO3, CH3CN, reflux; c) SnCl2·2H2O, EtOAc; d) S-(2- naphthylmethyl)-2-pyridylthioimidate·HBr, CH3CN/EtOH (1:3), rt.

Scheme 3.4 shows the synthesis of AIA azole hybrids 3.20a-c bearing alkoxy groups meta to the imidamide moiety. Protection of the 1-hydroxy group of 4-nitrocatechol was achieved according to a previously published procedure using MOMCl [167] followed by nucleophilic substitution of the desired alkyl iodide with MOM protected nitro phenol under basic condtions, yielding compounds 3.12a-c. Deprotection under acidic conditions followed by reaction with dibromooctane under basic conditions in dry acetonitrile provided 3.15a-c. Additional steps followed Scheme 3.1 to yield target compounds 3.20a-c.

Scheme 3.4: Synthesis of 3.20a-c. Reagents and conditions: a) MOMCl, K2CO3, DMF, 30ºC; b)

RI, K2CO3, CH3CN, sealed tube, 80ºC; c) HCl, MeOH/CH2Cl2, rt; d) 1,8-dibromooctane, K2CO3,

· CH3CN, reflux; e) imidazole, K2CO3, CH3CN, reflux; f) SnCl2 2H2O, EtOAc, reflux; g) S-(2- naphthylmethyl)-2-pyridylthioimidate·HBr, CH3CN/EtOH (1:3), rt.

Scheme 3.5 shows the synthesis of AIA azole hybrids 3.27a-c with alkoxy groups ortho to the imidamide moiety. Nucleophilic substitution of alkyl iodide by the hydroxyl group of 3.21 followed by nucleophilic aromatic substitution using sodium hydroxide was achieved according to a previously published procedure [168] yielding compounds 3.23a-c. Further steps followed the general synthetic strategy shown in Scheme 3.1 to yield final compounds 3.27a-c.

Scheme 3.5: Synthesis of 3.27a-c. Reagents and conditions: a) RI, K2CO3, sealed tube, DMF, 80

º C; b) NaOH, DMSO, reflux; c) 1,8-dibromooctane, K2CO3, CH3CN, reflux; d) imidazole,

. K2CO3, CH3CN, reflux; e) SnCl2 2H2O, EtOAc, reflux; f) S-(2-naphthylmethyl)-2- pyridylthioimidate·HBr, CH3CN/EtOH (1:3), rt.

Scheme 3.6 shows the synthesis of AIA azole hybrids 3.33a-j with either an unsubstituted biphenyl group or a substituted biphenyl with methoxy or isopropoxy group on the northern ring.

Nucleophilic substitution by 4-hydroxyphenylboronic acid pinacol ester 3.28 on dibromobutane or dibromohexane in dry acetonitrile under basic conditions yielded compounds 3.29a,b followed by nucleophilic attack by imidazole to give compounds 3.30a,b. Suzuki coupling of

3.30a,b with 4-nitroiodobenzene or 4-bromo-2-alkoxynitrobenzene using Pd[P(Ph)3]4 as a catalyst afforded compounds 3.31a-f [147]. Suzuki coupling reaction of 3.30a,b with 1-bromo-2- alkoxynitrobenzene using Pd(dppf)Cl2 as a catalyst gave compounds 3.31g-j [147]. Tin chloride reduction of 3.31a-j followed by reaction of the arylamine with the thioimidate salt gave final compounds 3.33a-j.

Scheme 3.6: Synthesis of 3.33a-j. Reagents and conditions: a) dibromoalkane, K2CO3, CH3CN, reflux; b) imidazole, K2CO3, CH3CN, reflux; c) 4-nitroiodobenzene or 4-bromo-2-methoxy-1- nitrobenzene or 4-bromo-2-isopropoxy-1-nitrobenzene, Pd[P(Ph)3]4, K2CO3, DMF, 100°C; d) 1- bromo-2-methoxy-4-nitrobenzene or 1-bromo-2-isopropoxy-4-nitrobenzene, Pd(dppf)Cl2,

K2CO3, DMSO, 100°C; e) SnCl2·2H2O, EtOAc, reflux; f) S-(2-naphthylmethyl)-2- pyridylthioimidate·HBr, CH3CN/EtOH (1:3), rt.

Scheme 3.7 shows the synthesis of AIA azole hybrids 3.42a-d containing a methoxy or isopropoxy group substituted on the southern ring of the biphenyl moiety ortho to alkoxyimidazole chain. Tosyl protection of 3.34 was achieved under basic conditions followed by the Miyaura borylation reaction of 3.35 yielding compound 3.36. Suzuki coupling reaction between 4-nitroiodobenzene and 3.36 led to the formation of compound 3.37a [147].

Compound 3.37b was prepared firstly by demethylation of 3.37a with boron tribromide followed by nucleophilic attack on isopropyl iodide under basic conditions. Deprotection of the tosyl group was achieved by heating 3.37a,b to reflux in 2N aq. NaOH [169] followed by reaction with the desired dibromoalkane to afford 3.39a-d. Nucleophilic attack by imidazole on 3.39a-d yielded compounds 3.40a-d. Tin chloride reduction of the nitro group and reaction of the resultant arylamines with the thioimidate salt was achieved to yield target products 3.42a-d.

Scheme 3.7: Synthesis of 3.42a-d. Reagents and conditions: a) TsCl, acetonitrile, K2CO3, reflux;

o b) bis(pinacolato)diboron, Pd(dppf)Cl2, AcOK, dioxane, 100 C; c) 1-iodo-4-nitrobenzene,

o o Pd[P(Ph)3]4 or Pd(dppf)Cl2, K2CO3, DME:DMF:H2O (7:3:1), 100 C: d) BBr3, DCM, 0 C; e) isopropyl iodide, K2CO3, acetonitrile; f) aq NaOH, EtOH/DMSO; g) dibromoalkane, acetonitrile,

K2CO3, reflux; h) imidazole, K2CO3, reflux; i) SnCl2·2H2O, ethyl acetate, reflux; j) S-(2- naphthylmethyl)-2-pyridylthioimidate·HBr, CH3CN/EtOH (1:3), rt.

Scheme 3.8 shows the synthesis of AIA azole hybrids 3.51a-f. Analogs 3.51a-d have methoxy or isopropoxy substitution on the southern ring meta to the alkoxyimidazole chain while analogs

3.51e,f have methoxy or isopropoxy substitution at the 3 and 3' position of the the biphenyl ring system. Tosyl protection of the 4-hydroxy group of 3.43 followed by reaction of the product with methyl iodide was achieved according to a previously published procedure yielding compound

3.44 [170]. Miyaura borylation of 3.44 followed by Suzuki coupling reaction with 4- nitroiodobenzene or 1-bromo-2-methoxy-4-nitrobenzene yielded 3.46a or 3.46c, respectively

[147]. Compounds 3.46b and 3.46d were prepared firstly by demethylation of 3.46a and 3.46c with boron tribromide followed by nucleophilic attack by the corresponding products on isopropyl iodide under basic conditions. Deprotection of the tosyl group was achieved by heating

3.46a-d to reflux with 2N aq. NaOH similar to a previously published procedure [169] followed by reaction of this product with dibromoalkane to afford 3.48a-f. Nucleophilic attack by imidazole yielded compounds 3.49a-f. Tin chloride reduction of the nitro group and reaction of the resulting arylamine with the thioimidate salt was achieved to afford target compounds 3.51a- f.

Scheme 3.8: Synthesis of 3.51a-f. Reagents and conditions: a) i)TsCl, acetonitrile, K2CO3; ii)

o CH3I, reflux; b) bis(pinacolato)diboron, Pd(dppf)Cl2, AcOK, dioxane, 100 C; c) 1-iodo-4- nitrobenzene or 1-bromo-2-methoxy-4-nitrobenzene, Pd(dppf)Cl2, K2CO3, DME:DMF:H2O

o o (7:3:1), 100 C: e) BBr3, DCM, 0 C; f) isopropyl iodide, K2CO3, DMF; g) aq NaOH,

EtOH/DMSO; h) dibromoalkane, acetonitrile, K2CO3, reflux; i) Imidazole, K2CO3, reflux; j)

SnCl2·2H2O, ethyl acetate, reflux; k) S-(2-naphthylmethyl)-2-pyridylthioimidate·HBr,

CH3CN/EtOH (1:3), rt.

3.3. Biological data

3.3.1 In vitro antileishmanial activity

IC50 and CC50 determinations were carried out by Dr. April Joice, Emilia Zywot, Heidi Meeds,

Xiaoping Liao, and Yena Kim. The evaluation of biological activity is ongoing for both the phenoxyalkyl and biphenyl AIA azole hybrids. Regarding phenoxyalkyl hybrids (Table 3.1 and

Table 3.2), the effect of three factors were evaluated: 1) chain length, 2) modification of the terminal groups, and 3) substitution with alkoxy or chloro groups on the phenoxy ring.

Homologation led to improvement in the antileishmanial activity as well as led to increased toxicity against both J774 macrophages and HepG2 cells. The optimal activity was obtained with an octyl chain in analog 3.12h with an IC50 value of 1.0 ± 0.1 μM against L. donovani intracellular amastigotes and CC50 value of 11 ± 3 μM and 12 ± 4 μM against J774 macrophages and HepG2 cells, respectively. Further increase in the chain length to ten carbons as in analog

3.12j did not lead to an improvement in the antileishmanial activity and only led to an increase in cytotoxicity with a CC50 value of 5.0 ± 1.6 μM against J774 macrophages. Regarding the terminal groups, imidazole as the azole moiety showed the best antileishmanial activity as mentioned for compound 3.12h earlier. Replacement of imidazole with triazole led to a 13-fold decrease in antileishmanial activity (compare compound 3.12h with compound 3.12i, which displays an IC50 value of 13 ± 2 μM against L. donovani intracellular amastigotes). However,

3.12i showed lower toxicity against J744 macrophages and HepG2 cells (CC50 values of 36 and

35 μM, respectively). Replacement of imidazole with pyrrole had little effect on antileishmanial and cytotoxicity results (compare data for 3.12l to 3.12h). Regarding the arylimidamide moiety,

74 replacing the pyridyl ring with a phenyl ring (analog 3.9k) led to a 22-fold and a 2.4-fold increase in cytotoxicity to J774 macrophages and HepG2 cells, respectively, compared to 3.12h.

Finally substitution of an alkoxy group on the phenoxy ring meta to the arylimidamide moiety didn’t lead to any improvement in the antileishmanial activity compared to 3.12h. However, placement of an isopropoxy group on the phenyl ring ortho to the imidamide terminal group increased the antileishmanial activity. Thus, analog 3.27c shows sub-micromolar antileishmanial activity, over 10-fold and 20-fold selectivity towards the parasite compared to J774 macrophages and HepG2 cells, respectively.

Table 3.1: In vitro antileishmanial activity and toxicity of 3.12a-la

Compound IC50 against L. CC50 against J774 CC50 against donovani (μM)b macrophages HepG2 (μM)b (μM)b 3.12a >50 >50 >50 3.12b >100 >50 >100 3.12c >25 >50 >50 3.12d 18 ± 5 >50 32 ± 6 3.12e >100 >50 >100 3.12f 13 ± 10 >50 42 ± 7 3.12g 12 ± 6 >40 20 ± 6 3.12h 1.0 ± 0.1 11 ± 3 12 ± 4 3.12i 13 ± 2 36 ± 2 35 ± 9 3.12j 1.4 ± 0.6 4.9 ± 1.6 6.9 ± 1.7 3.12k 1.2 ± 0.2 0.51 ± 0.03c 4.9 ± 1.9 3.12l 1.7 ± 0.5 11 ± 1 13 ± 4 a For standard compounds, an IC50 value of 0.044 ± 0.020 µM (n=58) was determined for amphotericin B against L. donovani, a CC50 value of 0.026 ± 0.006 µM (n=41) was determined for podophyllotoxin against J774 macrophages, and CC50 values of 0.18 ± 0.02 µM (n=38) for doxorubicin and 4.3 ± 1.7 µM (n=16) for quinacrine were determined, respectively, against HepG2 cells. All values for the standards represent the mean ± standard deviation. bMean ± standard deviation of at least three independent measurements cMean ± range of two independent measurements

Table 3.2: In vitro antileishmanial activity and toxicity of 3.12m,n, 3.20a-c and 3.27a-ca

Compound IC50 against L. CC50 against J774 CC50 against donovani (μM)b macrophages HepG2 (μM)b (μM)b 3.12m 1.3 ± 0.5 6.8 ± 1.3 7.7 ± 2.7 3.12n 2.1 ± 0.4 8.6 ± 2.1 8.6 ± 2.2 3.20a 2.2 ± 0.6 16 ± 0c 23 ± 4 3.20b 1.9 ± 0.5 9.0 ± 2.6 13 ± 0 3.20c 0.97 ± 0.12 7.9 ± 0.7 18 ± 2 3.27a 1.5 ± 0.8 In progress 16 ± 4 3.27b 0.73 ± 0.30 In progress 12 ± 3 3.27c 0.58 ± 0.06 6.7 ± 0.3 12 ± 3 a For standard compounds, an IC50 value of 0.044 ± 0.020 µM (n=58) was determined for amphotericin B against L. donovani, a CC50 value of 0.026 ± 0.006 µM (n=41) was determined for podophyllotoxin against J774 macrophages, and CC50 values of 0.18 ± 0.02 µM (n=38) for doxorubicin and 4.3 ± 1.7 µM (n=16) for quinacrine were determined, respectively, against HepG2 cells. All values for the standards represent the mean ± standard deviation. bMean ± standard deviation of at least three independent measurements cMean ± range of two independent measurements

Concerning the biphenoxyalkyl AIA azole hybrids, some compounds are still undergoing biological evaluation to give a better understanding of the structure-activity relationship. The current data show that biphenoxyalkyl analogs are generally more potent as well as more toxic against intracellular L. donovani amastigotes and J774 macrophages. The parent butyl analog

3.33a was much less active than its hexyl counterpart 3.33b with IC50 values of 2.6 ± 0.6 μM and

0.99 ± 0.31 μM, respectively. Regarding the northern ring substitutions (substitutions occurring on the phenyl ring bound to the imidamide group), the general trend is that the increase in chain length from butyl to hexyl or substitution with isopropoxy group instead of a methoxy group led to a slight improvement in biological activity. As shown in Table 3.3, all analogs except 3.33g,

3.33h and 3.33i displayed submicromolar antileishmanial potency. These compounds all possess an alkoxy substituent at the position meta to the arylimidamide group, which might indicate that an alkoxy group is disfavored at this position. However, analog 3.33j bearing a hexyl chain and substituted with isopropoxy group at the same position as 3.33g, 3.33h and 3.33i displayed the best antileishmanial activity in the biphenoxyalkyl AIA azole series. Southern ring substitutions

(substituents occurring on the phenyl ring bound to the alkoxy imidazole moiety, see Table 3.4) showed comparable activity to their northern ring counterparts, also generally following the trend of increased antileishmanial activity with increasing chain length. For the analogs possessing the alkoxy substituent ortho to the alkoxyimidazole group, more potent activity was observed with the isopropoxy substitution compared to the methoxy substitution (compare 3.42a to 3.42b and 3.42c to 3.42d). However, this order of potency was reversed when the alkoxy substituent was located meta to the alkoxyimidazole group, particularly for the analogs containing the six carbon linkers (compare 3.51c to 3.51d). In fact, compound 3.51c bearing a

78 methoxy substituent and a hexyl alkyl chain was the most potent analog in the southern analog series against intracellular L. donovani, displaying an IC50 value of 0.19 ± 0.03 μM. Finally, a preference for the bis-isopropoxy substitution compared to the bis-methoxy substitution was observed (compare 3.51e and 3.51f), but these compounds were both less potent than 3.33e,

3.33f, 3.33j, and 3.51c. Regarding cytotoxicity, biphenoxyalkyl AIA azole are slightly more toxic than their phenoxyalkyl counterparts especially against HepG2 cells. 3.33d showed less than 5 fold selectivity against the the parasite compared to J774 macrophages and less than 10 fold selectivity compared to HepG2 cells. Also, a rise in toxicity was observed by increasing chain length from butyl to hexyl except data for 3.33a and 3.33b (compare data of 3.33c with

3.33f) or by replacing the methoxy substituent with an isopropoxy group (compare data of 3.33c with 3.33d). 3.51a and 3.51c were considered the most promising compounds with more than 20 fold selectivity towards the parasite compared to J774 macrophages especially 3.51c with very good selectivity against L. donovani amastigotes compared to HepG2 cells. In conclusion, Figure

3.4 provides SAR studies to summarize the structure activity realationship of synthesized hybrids.

Table 3.3: In vitro antileishmanial activity and toxicity of 3.33a-ja

Compound IC50 against L. CC50 against J774 CC50 against donovani (μM)b macrophages HepG2 (μM)b (μM)b 3.33a 2.6 ± 0.6 13 ± 0 10 ± 1 3.33b 0.99 ± 0.31 9.5 ± 6.0 >25 3.33c 0.82 ± 0.25 7.6 ± 0.8c 13 ± 4 3.33d 0.60 ± 0.25 3.3 ± 1.8 4.5 ± 0.4c 3.33e 0.33 ± 0.13 5.1 ± 1.3c 8.5 ± 1.8 3.33f 0.29 ± 0.04 3.0 ± 0.3c 3.8 ± 0.5c 3.33g 2.2 ± 0.6 9.8 ± 0.2 16 ± 6 3.33h 1.5 ± 0.5 4.7 ± 0.7 7.7 ± 1.2 3.33i 1.2 ± 0.6 6.7 ± 0.7 In progress 3.33j 0.15 ± 0.04 3.4 ± 0.5 6.5 ± 0.4 a For standard compounds, an IC50 value of 0.044 ± 0.020 µM (n=58) was determined for amphotericin B against L. donovani, a CC50 value of 0.026 ± 0.006 µM (n=41) was determined for podophyllotoxin against J774 macrophages, and CC50 values of 0.18 ± 0.02 µM (n=38) for doxorubicin and 4.3 ± 1.7 µM (n=16) for quinacrine were determined, respectively, against HepG2 cells. All values for the standards represent the mean ± standard deviation. bMean ± standard deviation of at least three independent measurements cMean ± range of two independent measurements

Table 3.4: In vitro antileishmanial activity and toxicity of 3.42a-d and 3.51a-fa

Compound IC50 against L. CC50 against J774 CC50 against donovani (μM)b macrophages HepG2 (μM)b (μM)b 3.42a 3.0 ± 1.3 7.0 ± 1.9 10 ± 0c 3.42b 1.4 ± 0.9 4.7 ± 1.2 4.5 ± 1.1 3.42c 1.2 ± 0.4 3.9 ± 0.7 5.0 ± 1.0c 3.42d 0.69 ± 0.28 3.2 ± 0.3 4.8 ± 1.1 3.51a 0.46 ± 0.06 10 ± 3 >10 3.51b 0.78 ± 0.16 4.2 ± 0.3 In progress 3.51c 0.19 ± 0.03 4.3 ± 1.5 >100 3.51d 0.77 ± 0.13 3.1 ± 0.6c 5.9 ± 1.4 3.51e 0.94 ± 0.29 4.0 ± 0.5 7.4 ± 1.2 3.51f 0.49 ± 0.21 5.0 ± 0.9 6.2 ± 1.5 a For standard compounds, an IC50 value of 0.044 ± 0.020 µM (n=58) was determined for amphotericin B against L. donovani, a CC50 value of 0.026 ± 0.006 µM (n=41) was determined for podophyllotoxin against J774 macrophages, and CC50 values of 0.18 ± 0.02 µM (n=38) for doxorubicin and 4.3 ± 1.7 µM (n=16) for quinacrine were determined, respectively, against HepG2 cells. All values for the standards represent the mean ± standard deviation. bMean ± standard deviation of at least three independent measurements cMean ± range of two independent measurements

Figure 3.4: SAR trends for AIA azole hybrids

3.3.2. In vitro metabolic stability

Table 3.5 shows the metabolic stability of selected phenoxy alkyl or biphenoxyalkyl AIA azole hybrids. Regarding the phenoxyalkyl series, human microsomal stability testing showed that at substrate concentration of 1 µM, 3.20c was the least metabolically stable with t1/2 = 25 minutes, though it is metabolically stable at 3 µM concentration with 80% remaining after 60 minutes.

This might suggest that the compound inhibits its own metabolism at higher concentration or that the metabolic enzymes are saturated at higher concentration. 3.12j is also unstable in human liver

82 microsomes with t1/2 of 39 minutes and 57 minutes at 1 µM and 3 µM concentrations, respectively. Regarding stability when incubated with mouse liver microsomes, 3.12h and 3.12j were the least metabolically stable compounds with t1/2 of 16 minutes and 68 minutes for 3.12h and 20 minutes and 68 minutes for 3.12j at 1 µM and 3 µM concentrations, respectively. The rest of the phenoxyalkyl series analogs displayed better metabolic stability in both human and mouse liver microsomes (t1/2 > 60 minutes).

The biphenoxyalkyl AIA azole hybrids in human liver microsomes are generally more stable than their phenoxyalkyl counterparts, particularly at a substrate concentration of 1 µM. 3.51c is the least metabolically stable analog with > 70% of the compound remaining after 1 hour in experiments performed at 1 µM substrate concentration. Unlike other AIA azole analogs, testing of 3.51c at 3 µM concentration showed that > 60% of the compound was recovered after 60 minutes. Whether the compound is truly less stable at higher substrate concentrations is not known. Regarding experiments with mouse liver microsomes conducted at 1 µM substrate concentration for 3.33j and 3.51c show that their t1/2 was 34 minutes and 32 minutes, respectively. Experiments conducted at substrate concentrations of 3 µM have shown that 3.33c displayed a t1/2 of 65 minutes which is similar to its t1/2 at 1 µM substrate concentration (81 minute).

Table 3.5: Metabolic stability of selected AIA azole hybids

Human Liver Microsomal half-life (min) Mouse Liver Microsomal half-life (min) Compound Substrate Concentration Substrate Concentration 1 µM 3 µM 1 µM 3 µM 3.12h 79 min 100% after 60 min 16 min 70 min 3.12j 39 min 57 min 20 min 68 min 3.20a >90% after 60 min 100% after 60 min 100% after 60 min 100% after 60 min 3.20c 25 min >80% after 60 min 100% after 60 min 100% after 60 min 3.27a 150 min >80% after 60 min 120 min >80% after 60 min 3.27b 87 min >80% after 60 min >80% after 60 min 100% after 60 min 3.33c 70 min >80% after 60 min 81 min 65 min 3.33j >80% after 60 min >80% after 60 min 34 min 59 min 3.51a >80% after 60 min 100% after 60 min 100% after 60 min 100% after 60 min 3.51c >70% after 60 min >60% after 60 min 32 min 83 min

3.3.3. In vivo activity of 3.27c

Since 3.27c was the best phenoxyalkyl hybrid compound in the in vitro assays in terms of antileishmanial potency and selectivity, it was chosen for testing in L. donovani infected BALB/c mice. 3.27c was dissolved in 50% PEG 400 in water and was administered orally at doses of 37.5 mg/kg and 75 mg/kg for 5 days in groups of four infected animals. Miltefosine was used as a positive control and was administered orally at a dose of 10 mg/kg, also for five days. All animals survived the assay with less than 5% weight change in all tested animals. After quantitation of liver parasitemia, it was found that the antileishmanial activity was dose

84 dependent, with 17 ± 5% and 33 ± 7% reduction in liver parasitemia in the two treatment groups.

Treatment with miltefosine led to 95 ± 2% reduction of liver infection.

120 95 100

60 33

parasitemia 40 17

% Reduction in liverin Reduction% 20

0 37.5 mg/ kg 75 mg/kc 3.27c Miltefosine 3.27c

Figure 3.5: In vivo efficacy of 3.27c. Data are presented as the percentage reduction of liver

parasitemia compared to infected, untreated control animals. Bars and error bars indicate the

means and standard errors, respectively, of groups of four animals.

3.4 Conclusion

Joice et al. have shown that combination therapy of posaconazole and DB766 were synergistic in vivo due to possible improvement of pharmacokinetuic properties. Thus, development of hybrid

85 analogs possessing both the imidamide and an azole moiety might lead to the development of potent antileishmanial agents. A series of AIA azole hybrids with phenoxy alkyl or biphenoxyalkyl linkers were synthesized as antileishmanial candidates in this study. Compound

3.27c from the phenoxy alkyl series and compound 3.33j from the biphenoxyalkyl series were the most potent analogs against intracellular L. donovani amastigotes and also showed more than

10 fold selectivity towards the parasite compared to J774 macrophages and > 20-fold antileishmanial selectivity compared to HepG2 cells. Thus far, only 3.27c has been tested in L. donovani infected mice. The antileishmanial activity of 3.27c is dose dependent yet it is still inferior to both bis-AIA DB766 and mono-AIA DB2002, which might be attributed to several factors. Since microsomal metabolic stability studies have not yet been completed for 3.27c, the metabolic stability of 3.27c may impact its in vivo antileishmanial efficacy. Thus the use of another route as intraperitoneal administration to bypass the hepatic first pass effect might give an indication whether the low in vivo efficacy is due to metabolic effect or not. Testing of another potent compound might offer another alternative to this problem. Since 3.27a is stable in both human and liver microsomes and is only about 2 fold less potent than 3.27c, in vivo evaluation of this former compound is warranted. Since the mechanism of action of AIA azole hybrid analogs is unknown, there is a possibility that the azole moiety might not influence the biological activity and the attributed improvement in biological activity is due to increases in lipophilicity as reported for the phenoxyoctyl analog 3.12h compared to analogs with shorter alkyl chains. In support of this hypothesis, the biphenoxyhexyl analogs are generally more potent than than their butyl counterparts. Regarding the phenoxyalkyl series, 3.51a might be a good

86 candidate for in vivo studies as it displayed a submicromolar antileishmanail activity as well as it showed good metabolic stability in both human and mouse liver microsome studies.

Regarding future directions, further modification might be performed on both ends of AIA azole moieties as well as the linker moiety as shown in Figure 3.5. The use of a phenylalkyl linker (1) can be explored to determine the importance of the alkoxy group on antileishmanial activity, toxicity and lipophilicity. Synthesis of phenoxy or biphenoxyalkyl AIA analogs (2) without the azole might provide more information on the importance of the azole group. Also, in vitro results indicated that imidazole replacement with pyrrole led to minimal changes on both the antileishmanial activity as well as toxicity. Thus use of other nitrogen containing heterocycles (3) msuch as pyridine or pyrimidine may offer another alternative to azoles. Further replacement of the azole with tertiary amines, pyrrolidine or piperidine can be performed. Change of the imidamide terminal group from pyridyl to phenyl or other heterocycles was not well tolerated in the bis-AIA series. Thus synthesis of fused imidamide analogs (4) might provide an alternative approach to modify such a scaffold by restricting the rotation of the pyridyl group

Figure 3.6: Proposed structural modifications

Chapter 4

Experimental Section

Materials and Methods:

Unless otherwise noted, all reactions were carried out under nitrogen atmosphere in oven-dried glassware using standard syringe, cannula, and septa techniques. All commercial reagents, anhydrous solvents, and reagent-grade solvents were purchased from Sigma-Aldrich, Fisher

Scientific, VWR, and Combi-Blocks and used as received without further purification. Reactions were monitored by thin layer chromatography (TLC) using aluminum backed pre-coated silica gel plates from (TLC Silica Gel F-254, 200 μm, Dynamic Adsorbents). A UV lamp was used for visualization, as was ninhydrin and heat. Column chromatography was performed using silica gel

(60Å, pore size 63-200 μm, Sigma-Aldrich). Silica gel was deactivated by first washing with a

10% triethylamine solution in the eluent and then washing with the eluent itself (3x). Deuterated solvents for NMR were purchased from Sigma-Aldrich and used as received, with the exception of CDCl3. To prevent reactivity with acid sensitive compounds, the CDCl3 was stored over

K2CO3. NMR spectra were recorded on Bruker AV300, DRX400 or Bruker Ascend 700 MHz spectrometers and calibrated using the residual undeuterated solvent peak (CDCl3: δ 7.28 ppm

1 13 1 13 for H NMR, 77.02 ppm for C NMR; DMSO-d6: δ 2.50 ppm for H NMR, 40.02 ppm for C

NMR. Proton (1H) NMR data is reported as follows: chemical shift in ppm (multiplicity [as: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sex = sextet, sep = septet, m = multiplet, br = broad], coupling constant(s) in Hz, integration). Carbon (13C) NMR data was reported as chemical shift (δ) in ppm. Melting points were obtained on a Thomas Hoover Uni- melt capillary melting point apparatus. High resolution mass spectra (HRMS) were recorded on

Bruker microTOF (time-of-flight) mass spectrometer by electrospray ionization (ESI) time of flight experiments and reported as m/z.

4.1 Cyanines

Synthesis of methyl and methoxy substituted 4-methylquinolin-2(1H)-one (2.4a-g)

A mixture of 1 equivalent of ethyl acetoacetate (81.00-233 mmol) and 1 equivalent of aniline

(81-233 mmol) was heated in a 250 mL round bottom flask at 135-150 °C for 24-48 h [135]. The mixture was concentrated under vacuum and the crude substituted acetoacetamide derivatives were obtained either by precipitation from diethyl ether or by column chromatography using hexanes/ethyl acetate as eluent. The resulting methoxy or methyl substituted 3-oxo-N- phenylbutanamide was added to polyphosphoric acid (30 g) at 90-100 °C and heated in an oil bath at the same temperature for 2-3 h [136]. After the reaction was complete, the mixture was poured over ice and the pH was rendered neutral by adding 4N NaOH solution. The resulting precipitate was filtered and washed with water. The crude product was purified by column chromatography using ethyl acetate/hexanes as eluent to give the pure product as a white powder in 9-18% yield. For compounds 2.4a-g, the 1H NMR spectra obtained matched data from the literature [135, 136, 171]

4-Methylquinolin-2(1H)-one (2.4a) [136]. 3.3 g, yield 25% starting from 7.70 g aniline (82.9 mmol).

4,6-Dimethylquinolin-2(1H)-one (2.4b) [171]. 5.1 g, yield 13% starting from 24.20 g p-toluidine

(226.4 mmol).

4,7-Dimethylquinolin-2(1H)-one (2.4c) [171].3.5 g, yield 9% starting from 24.00 g m-toluidine

(224.5 mmol).

4,8-Dimethylquinolin-2(1H)-one (2.4d) [171]. 1.4 g, yield 9% starting from 9.62 g o-toluidine

(89.8 mmol)

6-Methoxy-4-methylquinolin-2(1H)-one (2.4e) [135]. 4.8 g, yield 13% starting from 24.00 g p- anisidine (195.1 mmol)

7-Methoxy-4-methylquinolin-2(1H)-one (2.4f) [135].3.0 g, yield 14% starting from 14 g m-

8-Methoxy-4-methylquinolin-2(1H)-one (2.4g) [135]. 6.30 g, yield 17%, starting from 22.80 g o- anisidine (185.0 mmol)

Synthesis of methyl and methoxy substituted 4-methylquinoline (2.5a-g)

Compounds 2.5a-g were prepared over 2 steps through a slight modification of a published procedure [139]. 2.4a-g (7.5-21.1 mmol) were added to excess POCl3 (15-25 mL) and the mixture was heated gently at 80 °C for 2 h. The temperature was increased to 120 °C and the reaction mixture was heated for an additional hour. Excess POCl3 was removed in vacuo, then the crude reaction mixture was poured on ice, basified with aqueous ammonium hydroxide solution to pH ≈ 10 and extracted with ethyl acetate (3 × 50 mL). The organic layers were combined, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue was purified by silica gel column chromatography to afford the product as a white powder in 83-98% yield. The

2-halogenated analogs were gently heated to reflux with 6 equivalents of 80% NH2NH2·H2O

(2.6-5.3 mL, 43.8-86.4 mmol) in absolute ethanol (15-25 mL) under a N2 atmosphere. 10% Pd/C

(98-170 mg, 0.9-1.6 mmol) was added in three portions over 2 hours and the reaction was monitored by TLC. After the disappearance of the starting material, the resulting suspension was filtered through celite while hot, then the filtrate was concentrated in vacuo. Water (30 mL) was added, then the crude product was extracted with DCM (3 × 50 mL) and dried over anhydrous

Na2SO4. The combined organic layers were evaporated in vacuo and the residue was purified by silica gel chromatography to afford the products 2.5a-g as white powders in 70-97% yield. For compounds 2.5a-g, the 1H NMR spectra obtained matched data from literature [172, 173].

4-Methylquinoline (2.5a) [172]. First step: Chromatography solvent: hexanes/ethyl acetate 5:1,

2.84 g, yield 85% starting from 3.00 g of 2.4a (18.84 mmol). Second step: Chromatography solvent: hexanes/ethyl acetate 3:1, 1.32 g, yield 84% starting from 2.00 g of 2-chloro-4- methylquinoline (11.25 mmol).

4,6-Dimethylquinoline (2.5b) [172]. First step: Chromatography solvent: hexanes/ethyl acetate

9:1, 1.90 g, yield 86 % strating from 2.00 g of 2.4b (11.54 mmol) Second step:

Chromatography solvent: hexanes/ethyl acetate 4:1, 1.28 g, yield 98% starting from 1.60 g of 2- chloro-4,6-dimethylquinoline (8.34 mmol).

4,7-Dimethylquinoline (2.5c) [172]. First step: Chromatography solvent: hexanes/ethyl acetate

7:1, 1.55 g, yield 70% starting from 2.00 g of 2.4c (11.54 mmol). Second step: Chromatography solvent: hexanes/ethyl acetate 4:1, 1.10 g, yield 76% starting from 1.8 g of 2-chloro-4,7- dimethylquinoline (9.39 mmol).

4,8-Dimethylquinoline (2.5d) [173]. First step: Chromatography solvent: hexanes/ethyl acetate

10:1, l.40 g, yield 97% starting 1.30 g 2.4d (7.50 mmol). Second step: Chromatography solvent: hexanes/ethyl acetate 3:1, 1.12 g, yield 98% starting from 1.40 g of 2-chloro-4,8- dimethylquinoline (7.30 mmol).

6-Methoxy-4-methylquinoline (2.5e) [172]. First step: Chromatography solvent: hexanes/ethyl acetate 6:1, 3.9 g, yield 89% starting from 4.00 g of 2.4e (21.14 mmol). Second step:

Chromatography solvent hexanes/ethyl acetate 4:1, 1.40 g, yield 86% starting from 2.00 g of 2- chloro-6-methoxy-4-methylquinoline (9.6 mmol).

7-Methoxy-4-methylquinoline (2.5f) [172]. First step: Chromatography solvent hexanes/DCM/ethyl acetate 12:1:0.5, 1.30 g, yield 59% starting 2.00 g of 2.4f (10.57 mmol)

Second step: Chromatography solvent: DCM/ethyl acetate 3:, 0.94 g, yield 56% starting from

2.00 g of 2-chloro-7-methoxy-4-methylquinoline (9.63 mmol).

8-Methoxy-4-methylquinoline (2.5g) [173]. First step: Chromatography solvent: hexanes/ethyl acetate 5:1, 4.20 g, yield 96% starting from 4.00 g of 2.4g (21.14 mmol). Second step:

Chromatography solvent: hexanes/ethyl acetate 4:1, 2.40 g, yield 96% starting from 3.00 g of 2- chloro-8-methoxy-4-methylquinoline (14.44 mmol).

Synthesis of 5-methyl or methoxy substituted 2-chloroaniline (2.8a,b)

To a solution of 4-substituted 1-chloro-2-nitrobenzene (29.1 mmol) in glacial acetic acid (60 mL) were added 5 equivalents of iron powder (8.13 g, 146 mmol) portion wise over a period of 30 min. The mixture was then heated at 100 °C for 1 h and the progress of the reaction was monitored by TLC. Upon completion of the reaction, the mixture was cooled to room temperature and filtered to remove the iron residue. This residue was washed with ethyl acetate

(2 × 100 mL), brine (100 mL) and water (100 mL), then the aqueous layer was extracted with ethyl acetate (3 × 75 mL). The organic layer was concentrated under reduced pressure, basified with 4N NaOH to pH ≈ 11 and partitioned with ethyl acetate (3 × 75 mL). The organic extracts were combined and washed with brine (100 mL) and water (100 mL) then dried over anhydrous

Na2SO4. The solvent was removed under reduced pressure and the crude product was subjected to silica gel chromatography employing DCM/hexanes 1:1 as the eluent to afford the product as a white powder in 83-85% yield [140]. Compounds 2.5a-g 1H NMR spectra obtained matched data from the literature [140, 174].

2-Chloro-5-methylaniline (2.8a) [174]. 3.42 g, yield 83%.

2-Chloro-5-methoxyaniline (2.8b) [140]. 3.90 g, yield 85%.

Synthesis of 5-methyl and 5-methoxy substituted 8-chloro-4-methylquinoline (2.10a, b)

Compounds 2.10a and 2.10b were prepared according to previously published procedure [142].

Concentrated sulfuric acid (3.6-4.0 mL) was added dropwise to a solution of the substituted aniline (2.8a or 2.8b) (25.4-28.2 mmol) in dioxane (20 mL) at room temperature. The mixture was gently heated to reflux and 1.5 equivalents of methyl vinyl ketone 2.9 (3.1-3.4 mL, 38.1-

42.3 mmol) in dioxane (10 mL) was added over a period of 3 hours. The reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated under reduced pressure and basified with saturated sodium carbonate to pH ≈ 9-10 and extracted with DCM (3 × 100 mL).

The combined organic layers were dried over anhydrous Na2SO4 and the crude product was purified by silica gel column chromatography to afford the pure chloro-substituted lepidine in

31-35% yield.

8-Chloro-4,5-dimethylquinoline (2.10a). Chromatography eluent: hexanes/ethyl acetate 7:1.

Yellow powder, 1.7 g, yield 31% starting from 4.00 g 2.8a (28.2 mmol); 1H NMR (300 MHz,

DMSO-d6) δ 2.86 (s, 3H), 2.91 (s, 3H), 7.35 (dd, J = 7.7, 0.6 Hz, 1H), 7.41 (dd, J = 4.2, 0.6 Hz,

13 1H), 7.78 (d, J = 7.7 Hz, 1H), 8.77 (d, J = 4.2 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 24.99,

25.32, 125.54, 129.04, 129.53, 129.94, 131.86, 136.17, 145.30, 146.95, 150.38.

8-Chloro-5-methoxy-4-methylquinoline (2.10b). Chromatography eluent: hexanes/isopropanol/ acetic acid 5:0.1:0.02. Yellow powder, 2.00 g, yield 38% starting from 4.00 g, 2.8b (25.4 mmol);

1 H NMR (300 MHz, CDCl3) δ 2.88 (d, J = 0.6 Hz, 3H), 3.94 (s, 3H), 6.76 (d, J = 8.5 Hz, 1H),

7.18 (dd, J = 4.4, 0.6 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 8.81 (d, J = 4.4 Hz, 1H); 13C NMR (75

MHz, CDCl3) δ 24.69, 55.63, 105.04, 122.25, 124.04, 125.08, 128.68, 145.76, 146.55, 150.47,

156.81.

Synthesis of 5-methyl and 5-methoxy substituted 4-methylquinoline (2.5h,i)

Compounds 2.5h and 2.5i were prepared following a previously published procedure [139]. 2.9a or 2.9b (8.60 mmol) was gently heated to reflux with 5 equivalents of 80% NH2NH2.H2O (2.62 mL, 43.0 mmol) in absolute ethanol (20 mL) under N2 atmosphere. 10% Pd/C (136 mg, 1.3 mmol) was added in 3 portions over 2 hours. The reaction was monitored by TLC and after the

98 disappearance of the starting material the resulting suspension was filtered through celite while hot. The filtrate was then concentrated in vacuo. Water (30 mL) was added and the product was extracted with DCM (3 × 50 mL) and dried over anhydrous Na2SO4. The combined organic layers were evaporated in vacuo and the residue was purified by silica gel chromatography to afford the product in 74-90% yield.

4,5-Dimethylquinoline (2.5h). Chromatography eluent: toluene/acetonitrile 10:1, off white

1 powder, 1.00 g, yield 74%. H NMR (300 MHz, CDCl3) δ 2.89 (s, 3H), 2.90 (s, 3H), 7.16 (dd, J

= 4.4, 0.6 Hz, 1H), 7.32 (d, J = 7.1 Hz, 1H), 7.54 (dd, J = 8.3, 7.1 Hz, 1H), 7.96 (d, J = 8.3 Hz,

13 1H), 8.69 (d, J = 4.4 Hz, 1H); C NMR (75 MHz, CDCl3) δ 25.34, 25.60, 124.04, 128.44,

128.63, 129.11, 129.50, 135.58, 145.55, 149.45, 149.95.

5-Methoxy-4-methylquinoline (2.5i). Chromatography eluent: hexanes/ethyl acetate/triethylamine 5:1:0.2 to 4:1:0.2, off white powder, 1.38 g, yield 93%. 1H NMR (300

MHz, CDCl3) δ 2.90 (s,3H), 3.96 (s, 3H), 6.86 (d, J = 8.1 Hz, 1H), 7.11 (d, J = 4.4 Hz, 1H), 7.57

(dd, J = 8.1 Hz, 1H), 7.69 (dd, J = 8.1, 0.8 Hz, 1H), 8.68 (d, J = 4.4 Hz, 1H); 13C NMR (75 MHz,

CDCl3) δ 24.64, 55.48, 105.17, 121.04, 122.45, 123.16, 128.77, 145.80, 150.11, 150.28, 157.66.

Synthesis of chloro- and bromo-substituted 4-methylquinoline (2.11a-f)

Concentrated sulfuric acid (1.3 mL, 24.3 mmol) was added dropwise to a solution of the bromo- or chloro-substituted aniline (2.2h-m) (15.7 mmol) in dioxane (15 mL) at room temperature. The mixture was gently heated to reflux, then 1.5 equivalent of methyl vinyl ketone (9, 1.9 mL, 23.50 mmol) in dioxane (10 mL) was added over a period of 3 hours. The reaction was monitored by

TLC. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, adjusted to pH ≈ 9-10 by the addition of saturated sodium carbonate and extracted with

DCM (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4 and the crude product was purified by silica gel column chromatography to afford the pure chloro- substituted lepidine in 19-74% yield [142]. Compounds 2.5a-g 1H NMR spectra matched data from the literature [142, 173, 175-177].

6-Chloro-4-methylquinoline (2.11a) [173]. Chromatography solvent: hexanes/ethyl acetate 6:1 to

4:1, brownish green powder, 0.52 g, yield 19%.

100

7-Chloro-4-methylquinoline (2.11b) [173]. Chromatography solvent: hexanes/ethyl acetate 6:1 to 4:1, brown powder, 0.82 g, yield 30%.

8-Chloro-4-methylquinoline (2.11c) [175]. Chromatography solvent: hexanes/ethyl acetate 10:1 to 7:1, yellowish white powder, 2.00 g, yield 72%.

6-Bromo-4-methylquinoline (2.11d) [142]. Chromatography solvent: hexanes/ethyl acetate 9:1 to

6:1, yellow powder, 0.80 g, yield 23%.

7-Bromo-4-methylquinoline (2.11e) [176]. Chromatography solvent: hexanes/ethyl acetate 9:1 to 6:1, yellow powder, 0.39 g, yield 12%.

8-Bromo-4-methylquinoline (2.11f) [177]. Chromatography solvent: hexanes/ethyl acetate 9:1 to 6:1, brownish green powder, 1.16 g, yield 33%.

101

Synthesis of 8-bromo-4-methyl-5-nitroquinoline (2.13)

Concentrated sulfuric acid (2.5 mL) was added dropwise to compound 2.11f (1.0 g, 4.50 mmol) at 0 °C, then KNO3 was added (0.58 g, 5.70 mmol) portion wise. The reaction was stirred at 0 °C for 2 hours then allowed to warm to r.t. with further stirring for 20 hours. After the reaction was complete, the mixture was poured over ice/water, basified with 2N sodium carbonate to pH ≈ 9-

10, extracted with ethyl acetate (3 × 50 mL) and dried over anhydrous Na2SO4. The combined organic layers were evaporated in vacuo and the residue was purified by silica gel chromatography (hexanes/DCM 2:3) to afford compound 2.13 as a yellow powder, 0.66 g, yield

1 55%. H NMR (300 MHz, DMSO-d6) δ 2.50 (s, 3H), 7.72 (d, J = 4.3 Hz, 1H), 8.04 (d, J = 8.1

13 Hz, 1H), 8.33 (d, J = 8.1 Hz, 1H), 9.02 (d, J = 4.3 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ

19.31, 120.58, 123.48, 127.48, 130.02, 132.27, 142.68, 145.03, 147.30, 152.63.

102

Synthesis of 5-amino-4-methylquinoline (2.14)

Compound 2.14 was prepared in 2 steps following previously published procedures [139, 140].

To a solution of compound 2.13 (1.60 g, 6.0 mmol) in glacial acetic acid (20 mL) was added 5 equivalents of iron powder (1.67 g, 29.90 mmol) portionwise over a period of 30 min. The reaction mixture was then heated at 100 °C for 1 hour and the progress of the reaction was monitored by TLC. Upon completion of the reaction, the mixture was cooled to room temperature and filtered to remove the iron residue. The residue was washed with ethyl acetate (2

× 100 mL), brine (100 mL) and water (100 mL), then the aqueous layer was extracted with ethyl acetate (3 × 75 mL). The organic layer was concentrated under reduced pressure, basified with

4N NaOH to pH ≈ 11 and partitioned with ethyl acetate (3 × 75 mL). The organic extracts were combined and washed with brine (100 mL) and water (100 mL) and dried over anhydrous

Na2SO4. The solvent was removed under reduced pressure to afford the product (1.42 g, 100%) which was taken directly to the next step without further purification. Five equivalents of 80%

NH2NH2·H2O (1.8 mL, 30.0 mmol) were added to a solution of the 8-bromo-4-methylquinoline

(1.4 g, in absolute ethanol (15 mL) under N2 atmosphere. 10% Pd/C (95 mg, 0.9 mmol) was

103 added in 3 portions over 2 hours, then the reaction was gently refluxed until the disappearance of the starting materials. This reaction mixture was filtered through celite while hot. The filtrate was concentrated in vacuo, water (30 mL) was added, and the product was extracted with DCM (3 ×

50 mL) and dried over anhydrous Na2SO4. The combined organic layers were evaporated in vacuo to afford the pure product as a yellow powder, 0.85 g, yield 90%. 1H NMR (300 MHz,

CDCl3) δ 2.96 (s, 3H), 4.34 (br s, 2H), 6.73 (dd, J = 7.4, 1.0 Hz, 1H), 7.01 (d, J = 4.4 Hz, 1H),

7.42 (dd, J = 7.4, 8.3 Hz, 1H), 7.54 (dd, J = 8.3, 1.0 Hz, 1H), 8.62 (d, J = 4.4 Hz, 1H); 13C NMR

(75 MHz, CDCl3) δ 24.45, 112.02, 119.89, 121.04, 122.71, 129.32, 143.91, 144.70, 149.70,

150.50.

Synthesis of 5-halo-4-methylquinoline (2.11g,h)

5-Chloro-4-methylquinoline (2.11g). The compound was synthesized according to a published procedure with slight modifications [145]. Compound 2.14 (0.35 g, 2.2 mmol) was suspended in

4N HCl (5 mL) and cooled to -10 °C. 1.2 equivalent of NaNO2 (0.18 g, 2.64 mmol) dissolved in

H2O (3 mL) was added over a period of 15 minutes. After stirring the mixture at -10 °C for an additional 15 minutes, 1.3 equivalent of CuCl (0.27 g, 2.73 mmol) dissolved in 4 mL conc HCl

104 was slowly added over 15 minutes and the mixture was allowed to warm to rt over 3 hours. The mixture was basified using 6N NH4OH, extracted with DCM and washed with water and brine.

The organic layer was dried over Na2SO4, evaporated in vacuo and purified by silica gel column chromatography (hexanes/ethyl acetate 3:1 to 1:1) to provide 2.11g as a yellow powder, 0.23 g,

1 yield 59%. H NMR (300 MHz, CDCl3) δ 3.09 (s, 3H), 7.23 (dd, J = 4.4, 0.7 Hz, 1H), 7.55 (dd, J

= 8.0, 7.6 Hz, 1H), 7.61 (dd, J = 7.6, 1.7 Hz, 1H), 8.04 (dd, J = 8.0, 1.7 Hz, 1H), 8.73 (d, J = 4.4

13 Hz, 1H); C NMR (75 MHz, CDCl3) δ 25.22, 125.17, 126.60, 128.40, 129.20, 130.02, 131.01,

145.39, 150.21, 150.28.

5-Bromo-4-methylquinoline (2.11h). The compound was synthesized following a general procedure given previously with slight modifications [146]. Compound 2.14 (0.30 g, 1.89 mmol) was suspended in 48% HBr (2 mL) and cooled to -10 °C. 1.14 equivalent of NaNO2 (0.15 g, 2.16 mmol) dissolved in H2O (2 mL) was added over a period of 15 minutes. The mixture was stirred at -10 °C for another 15 minutes, then 1.3 equivalents of CuBr (0.35 g, 2.43 mmol) dissolved in 4 mL 48% HBr/water 1:1 was slowly added over 15 minutes. The mixture was then allowed to warm to r.t. After stirring overnight, the mixture was basified with 2N Na2CO3 and extracted with ethyl acetate (3 × 50 mL). The organic layer was then dried over Na2SO4 and evaporated in vacuo. The crude product was purified by column chromatography (hexanes/ethyl acetate 3:1 to 1:1) to yield 2.11h as a yellow powder, 0.150 g, yield 36%. 1H NMR (300 MHz,

DMSO-d6) δ 3.07 (d, J = 0.7 Hz, 3H), 7.46 (dd, J = 4.4, 0.7 Hz, 1H), 7.59 (dd, J = 8.3, 7.6 Hz,

1H), 7.96 (dd, J = 7.6, 1.2 Hz, 1H), 8.04 (d, J = 8.3 and 1.2 Hz, 1H), 8.76 (d, J = 4.4 Hz, 1H);

13 C NMR (75 MHz, DMSO-d6) δ 25.20, 118.50, 126.05, 127.23, 129.91, 131.11, 133.98, 145.13,

150.12, 150.85.

105

Synthesis of phenyl substituted-4-methylquinolines (2.15a-d)

A three necked flask was charged with brominated quinoline compounds 2.11d-f or 2.11h (0.25 g, 1.1 mmol), 1.5 equivalents of phenylboronic acid (0.20 g, 1.68 mmol), 5 mol % Pd[(Ph)3P]4

(64 mg) and ethanol (10 mL). The flask was flushed with N2 gas, then 2 equivalents of N2 flushed 2N K2CO3 solution (0.30 g, 1 mL, 2.24 mmol) was added and the reaction was heated to reflux for 3 hours under N2 atmosphere. After completion of the reaction, the suspension was filtered over celite and the filtrate was concentrated under reduced pressure. Water (50 mL) was added and the mixture was partitioned with ethyl acetate (3 × 50 mL). The organic layer was dried over Na2SO4, evaporated in vacuo and the crude product was purified by silica gel column chromatography to yield 2.15a-d in 55-91% yield.

4-Methyl-5-phenylquinoline (2.15a). Chromatography eluent: DCM/ethyl acetate 96:4,

1 yellowish white powder, 0.22 g, yield 91%; H NMR (300 MHz, CDCl3) δ 2.03 (s, 3H), 7.14

(dd, J = 4.3, 0.6 Hz, 1H), 7.33-7.44 (m, 6H), 7.68 (dd, J = 8.4, 7.1 Hz, 1H), 8.15 (dd, J = 8.4, 1.3

13 Hz, 1H), 8.77 (d, J = 4.3 Hz, 1H); C NMR (75 MHz, CDCl3) δ 24.29, 124.29, 126.87, 127.25,

127.71, 127.79, 129.49, 129.53, 130.13, 140.45, 143.88, 145.57, 149.25, 149.78.

106

4-Methyl-6-phenylquinoline (2.15b). Chromatography eluent: DCM/acetonitrile: 96:4, colorless

1 oil, 0.22 g, yield 91%. H NMR (300 MHz, CDCl3) δ 2.77 (d, J = 0.5 Hz, 3H), 7.27-7.28 (m,

1H), 7.40-7.43 (m, 1H), 7.50-7.55 (m, 2H), 7.73-7.76 (m, 2H), 7.98 (dd, J = 8.6, 2.0 Hz, 1H),

13 8.17-8.20 (m, 2H), 8.79 (d, J = 4.3 Hz, 1H); C NMR (75 MHz, CDCl3) δ 18.75, 121.75,

122.24, 127.58, 127.67, 128.43, 128.87, 128.95, 130.48, 139.14, 140.83, 144.39, 147.38, 150.13.

4-Methyl-7-phenylquinoline (2.15c). Chromatography eluent: DCM/acetonitrile: 96:4, 0.14 g,

1 yield 57%. H NMR (300 MHz, CDCl3) δ 2.76 (s, 3H), 7.25 (d, J = 4.3 Hz, 1H), 7.43 (t, J = 7.3

Hz, 1H), 7.50-7.55 (m, 2H), 7.78-7.80 (m, 2H), 7.86 (dd, J = 8.7, 1.6 Hz, 1H), 8.09 (d, J = 8.7

13 Hz, 1H), 8.34 (d, J = 1.6 Hz, 1H), 8.82 (d, J = 4.4 Hz, 1H); C NMR (75 MHz, CDCl3) δ 18.61,

121.80, 124.35, 125.86, 127.39, 127.44, 127.62, 127.84, 128.98, 140.26, 141.79, 144.14, 148.36,

150.67

4-Methyl-8-phenylquinoline (2.15d) Compounds 2.15a 1H NMR spectra matched data from literature [178]. Chromatography eluent: DCM/hexanes 2:1, yellow powder, 0.13 g, yield 54%.

107

Synthesis of 1-substituted-4-methylquinolin-1-ium iodide (2.6a-l)

To a solution of compounds 2.5a-g (1.00 mmol) in methanol (3 mL) in a sealed tube was added excess methyl iodide (20.88 mmol, 1.3 mL) at room temperature. The sealed tube was heated at

80°C in an oil bath for 2-24 h. The solution became turbid with time, and after completion of the reaction, the sealed tube was allowed to cool to room temperature. Excess solution was then removed in vacuo. The precipitate was washed with benzene, filtered and dried to give the product in 42-97% yield. Compounds 2.6a-c 1H NMR spectra matched data from literature [149,

179].

1,4-Dimethylquinolin-1-ium iodide (2.6a) [149].Yellow powder, 0.26 g, yield 91%.

1-Ethyl-4-methylquinolin-1-ium iodide (2.6b) [179]. Yellow powder, 0.23 g, yield 77%.

1-Propyl-4-methylquinolin-1-ium iodide (2.6c) [179]. Yellow powder, 0.27 g, yield 86%.

108

1-Benzyl-4-methylquinolin-1-ium iodide (2.6d). Yellow powder, 0.28 g, yield 77%. 1H NMR

(300 MHz, DMSO-d6) δ 3.04 (s, 3H), 6.32 (s, 2H), 7.32-7.41 (m, 5H), 8.01 (ddd, J = 8.2, 7.0, 0.9

Hz, 1H), 8.15-8.21 (m, 2H), 8.46 (d, J = 9.0 Hz, 1H), 8.56 (dd, J = 8.4, 1.1 Hz, 1H), 9.60 (d, J =

13 6.1 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 20.34, 60.01, 120.14, 123.44, 127.58, 127.77,

129.16, 129.54, 129.67, 130.13, 134.54, 135.65, 137.34, 149.60, 160.10.

1,4,6-Trimethylquinolin-1-ium iodide (2.6e). 2.6e was further purified by silica gel chromatography (DCM/methanol 9.4:0.6) to give a light brown powder, 0.13 g, yield 43%. 1H

NMR (300 MHz, DMSO-d6) δ 2.65 (s, 3H), 2.97 (s, 3H), 4.55 (s, 3H), 8.00 (d, J = 5.9 Hz, 1H),

8.12 (d, J = 8.9 Hz, 1H), 8.32 (s, 1H), 8.38 (d, J = 8.9 Hz, 1H), 9.26 (d, J = 5.9 Hz, 1H); 13C

NMR (75 MHz, DMSO) δ 20.06, 21.51, 45.39, 119.73, 122.87, 125.93, 129.04, 136.63, 137.23,

140.52, 148.35, 157.69.

1,4,7-Trimethylquinolin-1-ium iodide (2.6f). Light yellow powder, 0.22 g, yield 73%. 1H NMR

(300 MHz, DMSO-d6) δ 2.70 (s, 3H), 2.97 (s, 3H), 4.53 (s, 3H), 7.91 (d, J = 8.6 Hz, 1H), 7.96

(d, J = 6.0 Hz, 1H), 8.30 (s, 1H), 8.42 (d, J = 8.6 Hz, 1H), 9.27 (d, J = 6.0 Hz, 1H); 13C NMR (75

MHz, DMSO-d6) δ 19.95, 22.42, 45.34, 118.88, 122.10, 126.97, 127.22, 132.00, 138.42, 146.97,

148.94, 158.22.

1,4,8-Trimethylquinolin-1-ium iodide (2.6g). 2.6g was further purified by silica gel chromatography (DCM/methanol 9.4:0.6) to afford a yellowish green powder, 0.23 g, yield 76%.

1 H NMR (300 MHz, DMSO-d6) δ 2.97 (s, 3H), 3.08 (s, 3H), 4.78 (s, 3H), 7.86-7.92 m, 1H), 8.00

(d, J = 6.0 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 8.35 (d, J = 7.6 Hz, 1H), 9.24 (d, J = 6.0 Hz, 1H);

109

13 C NMR (75 MHz, DMSO-d6) δ 20.83, 25.32, 51.14, 122.77, 125.88, 129.64, 130.80, 131.04,

139.21, 139.67, 151.36, 158.90.

6-Methoxy-1,4-dimethylquinolin-1-ium iodide (2.6h). 2.6h was further purified by silica gel chromatography (DCM/ethanol 9.2:0.8). Light yellow powder, 0.22 g, yield 70%. 1H NMR (300

MHz, DMSO-d6) δ 2.96 (s, 3H), 4.06 (s, 3H), 4.55 (s, 3H), 7.69 (d, J = 2.7 Hz, 1H), 7.90 (dd, J =

9.6, 2.7 Hz, 1H), 7.98 (d, J = 6.0 Hz, 1H), 8.42 (d, J = 9.6 Hz, 1H), 9.16 (d, J = 6.0 Hz, 1H); 13C

NMR (75 MHz, DMSO-d6) δ 20.25, 45.53, 56.85, 105.44, 121.80, 123.14, 127.03, 130.86,

133.75, 146.49, 156.57, 159.67.

7-Methoxy-1,4-dimethylquinolin-1-ium iodide (2.6i). Light yellow powder, 0.27 g, yield 86%.

1 H NMR (300 MHz, DMSO-d6) δ 2.94 (s, 3H), 4.11 (s, 3H), 4.50 (s, 3H), 7.63 (d, J = 2.2 Hz,

1H), 7.68 (dd, J = 9.2, 2.2 Hz, 1H), 7.84 (d, J = 6.1 Hz, 1H), 8.45 (d, J = 9.2 Hz, 1H), 9.17 (d, J

13 = 6.1 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 20.25, 45.53, 56.85, 105.44, 121.80, 123.14,

127.03, 130.86, 133.75, 146.49, 156.57, 159.67.

8-Methoxy-1,4-dimethylquinolin-1-ium iodide (2.6j). 2.6j was further purified by silica gel chromatography (DCM/methanol= 9.4:0.6). Off white powder, 0.14 g, yield 45%. 1H NMR (300

MHz, DMSO-d6) δ 2.93 (s, 3H), 4.08 (s, 3H), 4.73 (s, 3H), 7.77 (d, J = 7.6 Hz, 1H), 7.91-8.01

13 (m, 3H), 9.18 (d, J = 6.0 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 20.76, 52.28, 57.82, 116.36,

118.59, 123.26, 130.33, 130.62, 131.29, 150.93, 152.20, 157.97.

1,4,5-Trimethylquinolin-1-ium iodide (2.6k). Yellow powder, 0.27 g, yield 90%. 1H NMR (300

MHz, DMSO-d6) δ 3.04 (s, 3H), 3.19 (s, 3H), 4.54 (s, 3H), 7.85 (d, J = 7.2 Hz, 1H), 7.97 (d, J =

110

6.1 Hz, 1H), 8.09 (dd, J = 8.8, 7.2 Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H), 9.29 (d, J = 6.1 Hz, 1H);

13 C NMR (75 MHz, DMSO-d6) δ 25.55, 26.65, 46.43, 118.32, 124.67, 128.78, 129.51, 133.15,

134.62, 139.98, 148.42, 160.33.

5-Methoxy-1,4-dimethylquinolin-1-ium iodide (2.6l). Yellow-orange powder, 0.30 g, yield 95%.

1 H NMR (300 MHz, DMSO-d6) δ 3.06 (s, 3H), 4.06 (s, 3H), 4.48 (s, 3H), 7.48 (d, J = 8.1 Hz,

1H), 7.87 (d, J = 6.1 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 8.12-8.19 (m, 1H), 9.25 (d, J = 6.1 Hz,

13 1H); C NMR (75 MHz, DMSO-d6) δ 26.19, 46.11, 57.36, 110.00, 111.08, 121.75, 123.56,

136.36, 140.07, 148.76, 159.29, 159.49.

Synthesis of chloro substituted-4-methylquinolin-1-ium iodide (2.12a,b and 2.12d)

Compounds 2.12a,b and 2.12d were synthesized from 2.11a,b and 2.11d (1 mmol) following the general synthesis of quinolinium iodide salts 2.6a-l.

6-Chloro-1,4-dimethylquinolin-1-ium iodide (2.12a). 2.12a was further purified by silica gel chromatography (DCM/methanol 9.3:0.7). Orange powder, 0.21 g, yield 66%. 1H NMR (300

MHz, DMSO-d6) δ 2.99 (s, 3H), 4.57 (s, 3H), 8.10 (d, J = 6.0 Hz, 1H), 8.32 (dd, J = 9.4, 2.2 Hz,

111

1H), 8.53 (d, J = 9.4 Hz, 1H), 8.62 (d, J = 2.2 Hz, 1H), 9.38 (d, J = 6.0 Hz, 1H); 13C NMR (75

MHz, DMSO-d6) δ 20.18, 45.77, 122.49, 123.96, 126.24, 129.99, 135.00, 135.33, 136.94,

149.89, 158.17.

7-Chloro-1,4-dimethylquinolin-1-ium iodide (2.12b). 2.12b was further purified by silica gel chromatography (DCM/methanol 9.6/0.4 to 9.4/0.6). Brown powder, yield 75%, 0.24 g; 1H NMR

(300 MHz, DMSO-d6) δ 2.99 (s, 3H), 4.55 (s, 3H), 8.05 (dd, J = 6.0, 0.7 Hz, 1H ), 8.11 (dd, J =

9.0, 1.9 Hz, 1H), 8.55 (d, J = 9.0 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H), 9.34 (d, J = 6.0 Hz, 1H); 13C

NMR (75 MHz, DMSO-d6) δ 20.15, 45.79, 119.68, 123.35, 127.75, 129.39, 130.67, 138.85,

140.37, 150.35, 158.84.

5-Chloro-1,4-dimethylquinolin-1-ium iodide (2.12d) Greenish yellow powder, yield 53%, 0.17

1 g; H NMR (300 MHz, DMSO-d6) δ 3.27 (br s, 3H), 4.56 (s, 3H), 8.10 (d, J = 5.9 Hz, 1H), 8.14-

13 8.20 (m, 2H), 8.46-8.51 (m, 1H), 9.39 (d, J = 6.0 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ

26.79, 46.79, 120.07, 126.24, 127.75, 133.29, 133.35, 134.73, 140.54, 149.79, 158.90.

112

Synthesis of 8-chloro-1,4-dimethylquinolin-1-ium trifluoromethane-sulfonate (2.12c)

Compound 2.12c was prepared following a previously published procedure [143]. To a solution of 8-chloro-4-methyl quinoline (1.0 g, 5.62 mmol) in anhydrous toluene (3 mL) was added methyl trifluoromethanesulfonate (3.1 mL, 27.4 mmol) in a 25 mL round bottom flask. The solution was allowed to stir at rt for 24 h, then the solvent was removed under reduced pressure.

The residue was purified by silica gel chromatography (DCM/methanol 9.6:4) to afford the pure

1 product 2.9c as a buff powder, 1.30 g, yield 68%. H NMR (300 MHz, DMSO-d6) δ 3.00 (s, 3H),

4.88 (s, 3H), 7.97 (m, 1H), 8.11 (d, J = 6.0 Hz, 1H), 8.37 (d, J = 7.7 Hz, 1H), 8.51 (d, J = 8.3

13 Hz, 1H), 9.34 (d, J = 6.0 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 20.93, 52.05, 121.14 (q, J =

321 Hz) 123.80, 124.62, 127.44, 130.12, 132.01, 136.01, 138.98, 153.29, 159.92.

113

Synthesis of phenyl-substituted-4-methylquinolin-1-ium iodide (2.16a-d)

Compounds 2.16a-d were synthesized from 2.15a-d (1 mmol) following the general synthesis of quinolinium iodide salts 2.6a-l.

5-Phenyl-1,4-dimethylquinolin-1-ium iodide (2.16a). Yellow powder, 0.25 g, yield 69%. 1H

NMR (300 MHz, DMSO-d6) δ 2.20 (s, 3H), 4.63 (s, 3H), 7.38-7.42 (m, 2H), 7.52-7.57 (m, 3H),

7.83 (dd, J = 7.2, 1.0 Hz, 1H), 7.96 (d, J = 6.0 Hz, 1H), 8.25 (dd, J = 8.9, 7.2 Hz, 1H), 8.55 (dd, J

13 = 8.9, 1.0 Hz, 1H), 9.36 (d, J = 6.0 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 25.61, 46.37,

119.79, 125.35, 127.58, 128.95 (overlapped 2Cs), 129.60, 133.17, 134.01, 139.53, 141.52,

142.80, 148.75, 159.17.

6-Phenyl-1,4-dimethylquinolin-1-ium iodide (2.16b). Yellow powder, 0.27 g, yield 75%. 1H

NMR (300 MHz, DMSO-d6) δ 3.09 (s, 3H), 4.60 (s, 3H), 7.50-7.53 (m, 1H), 7.55-7.63 (m, 2H),

7.99-8.02 (m, 2H), 8.05 (m, d, J = 6.0 Hz, 1H), 8.55 (d, J = 9.1 Hz, 1H), 8.60 (dd, J = 9.1, 1.6

13 Hz, 1H), 8.67 (d, J = 1.6 Hz, 1H), 9.33 (d, J = 6.0 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ

114

20.24, 45.51, 120.77, 123.28, 124.12, 128.22, 129.43, 129.48, 129.76, 134.21, 137.54, 138.08,

141.35, 149.05, 158.85.

7-Phenyl-1,4-dimethylquinolin-1-ium iodide (2.16c). Yellow powder, 0.34 g, yield 94%. 1H

NMR (300 MHz, DMSO-d6) δ 3.02 (s, 3H), 4.67 (s, 3H), 7.54-7.66 (m, 3H), 8.01-8.07 (m, 3H),

8.40 (dd, J = 8.9, 1.4 Hz, 1H), 8.58-8.61 (m, 2H), 9.34 (d, J = 6.0 Hz, 1H); 13C NMR (75 MHz,

DMSO-d6) δ 20.02, 45.53, 116.96, 122.74, 128.01, 128.12, 128.53, 129.06, 129.82, 130.10,

138.10, 138.74, 146.53, 149.71, 158.31.

8-Phenyl-1,4-dimethylquinolin-1-ium iodide (2.16d). Yellow powder, 0.25 g, yield 69%. 1H

NMR (300 MHz, DMSO-d6) δ 3.06 (s, 3H), 3.86 (s, 3H), 7.49-7.57 (m, 5H), 8.00-8.08 (m, 2H),

8.10 (d, J = 6.0 Hz, 1H), 8.58 (dd, J = 7.5, 2.3 Hz, 1H), 9.22 (d, J = 6.0 Hz, 1H); 13C NMR (75

MHz, DMSO-d6) δ 20.80, 51.47, 123.23, 127.26, 129.04, 129.07, 129.89, 130.52, 134.80,

+ 136.95, 139.44, 140.59, 152.04, 159.18. ; HRMS (ESI) m/z M calcd for C17H16N, 234.12773; found, 234.12822

115

Synthesis of 3-methyl-2-(methylthio)benzothiazolium iodide (2.18a).

Compound 2.18a was synthesized from 2-(methylthio)benzothiazole following a previously published procedure 1H NMR spectra matched data from literature [21]. White powder, 2.85 g, yield 80%.

Synthesis of 3-methyl-2-(methylthio)benzothiazolium trifluoromethane-sulfonate (2.18b).

Compound 2.18b was synthesized following the same procedure as listed for 2.9c and was isolated as a white powder, 1.27 g, yield 67% (starting from 1.00 g 2-(methylthio)benzothiazole).

116

1 H NMR (300 MHz, DMSO-d6) δ 3.12 (s, 3H), 4.11 (s, 3H), 7.69-7.76 (m, 1H), 7.81-7.88 (m,

1H), 8.20 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H)

1-Alkyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium analogs (2.1a-t)

To a solution of 2.6a-l, 2.12a-d or 2.16a-d (0.73-0.30 mmol) and 1.1 equivalent of 3-methyl-2-

(methylthio)benzo[d]thiazol-3-ium iodide 2.18a or trifluoromethane-sulfonate 2.18b (0.33-0.80 mmol) in ethanol:DMF (1:1, 10 ml) was added 5 equivalents of triethylamine (0.28 mL, 2

117 mmol). Upon addition of triethylamine, instantaneous red color formation was observed. The reaction was heated gently in an oil bath at 80 °C overnight in the dark. After the reaction was completed, the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography then further recrystallized from DMF/water to yield the pure product as a red powder.

1-Methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.1a)

[149]: red powder; 98 mg, yield 65% starting from 2.6a (100 mg, 0.35 mmol); mp 275-276 °C;

1 H NMR (300 MHz, DMSO-d6) δ 3.98 (s, 3H), 4.14 (s, 3H), 6.87 (s, 1H), 7.31 (d, J = 7.2 Hz,

1H), 7.38 (brt, J = 7.5 Hz, 1H), 7.58 (brt, J = 6.9 Hz, 1H), 7.71-7.78 (m, 2H), 7.96-8.03 (m, 3H),

13 8.58 ( d, J = 7.2 Hz, 1H), 8.76 (d, J = 8.4 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 34.26,

42.84, 88.33, 108.22, 113.32, 118.67, 123.27, 124.24, 124.41, 124.85, 125.94, 127.35, 128.57,

+ 133.64, 138.43, 140.87, 145.44, 148.95, 160.20; HRMS (ESI) m/z M calcd for C19H17N2S,

305.11070; found, 305.11038.

1-Ethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.1b): red powder; 89 mg, yield 57% starting from 2.6b (105 mg, 0.35 mmol); mp 258-260 °C; 1H NMR

(300 MHz, DMSO-d6) δ 1.48 (t, J = 7.2 Hz, 3H) , 4.01 (s, 3H), 4.64 (q, J = 7.2 Hz, 2H), 6.91 (s,

1H), 7.37 (d, J = 7.2 Hz, 1H), 7.38-7.41 (m, 1H, overlapped), 7.60 (ddd, J = 8.4, 7.3, 1.2 Hz,

1H), 7.73-7.78 (m, 2H), 8.00 (ddd, J = 8.5, 7.3, 1.2 Hz, 1H) 8.04 (dd, J = 7.8, 0.6 Hz, 1H, overlapped) , 8.14 ( d, J = 8.7 Hz, 1H), 8.65 (d, J = 7.2 Hz, 1H), 8.79 (d, J = 8.0 Hz, 1H); 13C

NMR (75 MHz, DMSO-d6) δ 15.16, 34.31, 49.93, 88.49, 108.61, 113.43, 118.46, 123.34,

118

124.30, 124.73, 124.93, 126.33, 127.25, 128.62, 133.77, 137.33, 140.95, 144.47, 149.05, 160.43;

+ HRMS (ESI) m/z M calcd for C20H19N2S, 319.12635; found, 319.12615.

1-Propyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.1c): red powder; 100 mg, yield 62% starting from 2.6c (110 mg, 0.35 mmol); mp 265-266 °C; 1H NMR

(300 MHz, DMSO-d6) δ 0.95 (t, J = 7.3 Hz, 3H) , 1.89 (h, J = 7.3 Hz, 2H), 4.01 (s, 3H), 4.56 (t, J

= 7.3 Hz, 2H), 6.92 (s, 1H), 7.36 (d, J = 7.2 Hz, 1H), 7.41 (ddd, J = 7.9, 7.4, 1.0 Hz, 1H), 7.61

(ddd, J = 8.5, 7.3, 1.2 Hz, 1H), 7.73-7.79 (m, 2H), 7.98 (ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 8.04 (dd,

J = 7.9, 0.8 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.64 (d, J = 7.2 Hz, 1H) , 8.79 ( d, J = 7.9 Hz,

13 1H); C NMR (75 MHz, DMSO-d6) δ 11.14, 22.67, 34.32, 55.93, 88.57, 108.24, 113.45, 118.61,

123.35, 124.32, 124.70, 124.95, 126.30, 127.25, 128.63, 133.71, 137.51, 140.95, 144.88, 149.04,

+ 160.51; HRMS (ESI) m/z M calcd for C21H21N2S, 333.14194; found, 333.14192.

1-Benzyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.1d): red powder; 120 mg, yield 77%, starting from 2.6d (110 mg, 0.30 mmol); mp 271-273 °C; 1H NMR

(300 MHz, DMSO-d6) δ 4.06 (s, 3H), 5.88 (s, 2H), 6.98 (s, 1H), 7.29-7.41 (m, 5H), 7.43-7.48

(m, 2H), 7.62-7.72 (m, 2H), 7.82-7.90 (m, 2H), 7.98 (dd, J = 8.7, 0.9 Hz, 1H), 8.08 (dd, J = 8.0,

13 0.9 Hz, 1H), 8.78-8.82 (m, 2H); C NMR (75 MHz, DMSO-d6) δ 34.48, 57.27, 89.18, 108.25,

113.72, 118.96, 123.43, 124.54, 124.78, 125.21, 126.30, 127.09, 127.23, 128.63, 128.74, 129.47,

133.63, 135.94, 137.70, 140.96, 145.32, 149.16, 161.18; HRMS (ESI) m/z M+ calcd for

C25H21N2S, 381.14185; found, 381.14187.

1,5-Dimethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.1e).

Bright red powder; 95 mg, yield 32% starting from 2.6k (200 mg, 0.67 mmol); mp 271-273°C;

119

1 H NMR (300 MHz, DMSO-d6) δ 3.03 (s, 3H), 3.93 (s, 3H), 4.11 (s, 3H), 6.76 (s, 1H), 7.36-7.46

(m, 2H), 7.58-7.65 (m, 2H), 7.79-7.87 (m, 3H), 8.05 (dd, J = 8.0, 0.8 Hz, 1H), 8.49 (d, J = 7.1

13 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 21.78, 34.19, 43.27, 96.19, 109.01, 113.41, 116.18,

123.40, 124.30, 125.01, 128.66, 131.29, 132.42, 138.15, 140.34, 141.00, 144.14, 150.41, 159.36;

+ HRMS (ESI) m/z M calcd for C20H19N2S, 319.12635; found, 319.12599.`

1,6-Dimethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.6f):

Dark red powder; 160 mg, yield 49% starting from 2.6e (220 mg, 0.73 mmol); mp 289-291°C;

1 H NMR (300 MHz, DMSO-d6) δ 2.61 (s, 3H), 4.00 (s, 3H), 4.16 (s, 3H), 6.87 (s, 1H), 7.34 (d, J

= 7.1 Hz, 1H), 7.39 (td, J = 7.6, 1.0 Hz, 1H), 7.59 (ddd, J = 8.4, 7.3, 1.2 Hz, 1H), 7.74 ( d, J =

8.2 Hz, 1H), 7.85 (dd, J = 8.9, 1.4 Hz, 1H), 7.96 (d, J = 8.9 Hz, 1H), 8.01 (dd, J = 7.9, 0.8 Hz,

13 1H), 8.52-8.56 (m, 2H); C NMR (75 MHz, DMSO-d6) δ 21.32, 34.22, 42.88, 88.08, 108.38,

113.22, 118.59, 123.25, 124.15, 124.53, 124.73, 124.90, 128.54, 135.26, 136.74, 137.61, 140.99,

+ 144.88, 148.72, 159.085 HRMS (ESI) m/z M calcd for C20H19N2S, 319.12635; found,

319.12584.

1,7-Dimethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.1g):

Red crystals; 200 mg, yield 61% starting from 2.6f (220 mg, 0.73 mmol); mp 268-270°C; 1H

NMR (300 MHz, DMSO-d6 at 310 °K) δ 2.62 (s, 3H), 3.99 (s, 3H), 4.16 (s, 3H), 6.89 (s, 1H),

7.35 (d, J = 7.1 Hz, 1H), 7.40 (td, J = 7.6, 0.9 Hz, 1H), 7.56-7.65 (m, 2H), 7.74 (d, J = 8.0 Hz,

1H), 7.88 (s, 1H), 8.00 (dd, J = 8.1, 0.9 Hz, 1H), 8.57 (d, J = 7.1 Hz, 1H), 8.67 (d, J= 8.9 Hz,

13 1H); C NMR (75 MHz, DMSO-d6 at 335 °K ) δ 21.26, 33.47, 42.10, 87.45, 107.44, 112.46,

120

117.24, 121.94, 122.46, 123.48, 124.05, 125.07, 127.82, 128.37, 138.02, 140.32, 144.04, 144.59,

+ 148.56, 159.38; HRMS (ESI) m/z M calcd for C20H19N2S, 319.12635; found, 319.12586.

1,8-Dimethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (2.1h):

Red powder; 150 mg, yield 46% starting from 2.6g (220 mg, 0.73 mmol); mp 221-224 °C; 1H

NMR (300 MHz, DMSO-d6 at 310 °K) δ 2.92 (s, 3H), 3.98 (s, 3H), 4.37 (s, 3H), 6.84 (s, 1H),

7.35 (d, J = 7.2 Hz, 1H, overlapped), 7.40 (td, J = 7.6, 1.0 Hz, 1H, overlapped), 7.57- 7.64 (m,

2H), 7.75 (d, J = 8.4 Hz, 1H, overlapped), 7.8 (d, J = 7.2 Hz, 1H, overlapped), 8.02 (dd, J = 7.9,

13 1.0 Hz, 1H), 8.51 (d, J = 7.2 Hz, 1H), 8.60 (d, J = 8.0 Hz, 1H); C NMR (75 MHz, DMSO-d6 at

310 °K) δ 24.87, 34.24, 48.39, 88.89, 108.49, 113.29, 123.27, 124.21, 124.37, 124.84, 126.23,

127.02, 128.57, 129.48, 138.11, 139.62, 140.99, 147.86, 149.66, 160.27; HRMS (ESI) m/z M+ calcd for C20H19N2S, 319.12635; found, 319.12589.

5-Methoxy-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1i): Red crystals; 86 mg, yield 49% starting from 2.6l (120 mg, 0.38 mmol); mp 217-218 °C;

1 H NMR (300 MHz, DMSO-d6) δ 3.85 (s, 3H), 4.03 (s, 3H), 4.11 (s, 3H), 7.14 (d, J= 7.3 Hz,

1H), 7.28 (d, J = 8.1 Hz, 1H), 7.38 (td, J = 7.6, 0.9 Hz, 1H), 7.47 (dd, J = 8.7, 0.7 Hz, 1H), 7.57

(ddd, J = 8.4, 7.3, 1.2 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.86 (t, J= 8.4 Hz, 1H), 7.92 (s, 1H),

13 8.00 (dd, J = 8.0, 0.9 Hz, 1H), 8.42 (d, J = 7.3 Hz, 1H); C NMR (75 MHz, DMSO-d6 at 300

°K) δ 34.12, 43.47, 57.34, 95.00 108.66, 108.90, 110.11, 113.34, 115.70, 123.28, 124.30, 124.87,

128.59, 133.93, 140.78, 140.82, 144.16, 149.03, 159.47, 160.17; HRMS (ESI) m/z M+ calcd for

C20H19ON2S, 335.12126; found, 335.12088.

121

6-Methoxy-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1j): Bright red powder; 118 mg, yield 41% starting from 2.6h (200 mg, 0.63 mmol) mp 273-

1 275 °C; H NMR (300 MHz, DMSO-d6) δ 3.98 (s, 3H), 4.04 (s, 3H), 4.18 (s, 3H), 6.73 (s, 1H),

7.33-7.41 (m, 2H), 7.54-7.61 (m, 1H), 7.67-7.74 (m, 2H), 7.93 (d, J = 2.6 Hz, 1H), 7.99 (dd, J =

7.9, 0.9 Hz, 1H), 8.05 (d, J = 9.5 Hz, 1H), 8.55 (d, J = 7.2 Hz, 1H); 13C NMR (75 MHz, DMSO- d6 at 310 °K) δ 33.51, 42.53, 56.04, 87.49, 106.03, 107.90, 112.46, 120.05, 122.60, 122.76,

123.42, 124.01, 125.50, 127.89, 132.89, 140.45, 143.32, 147.66, 157.77, 158.86; HRMS (ESI)

+ m/z M calcd for C20H19ON2S, 335.12126; found, 335.12089.

7-Methoxy-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1k): Reddish brown powder; 95 mg, yield 32% starting from 2.6i (200 mg, 0.63 mmol); mp

1 271-273 °C; H NMR (300 MHz, DMSO-d6) δ 3.95 (s, 3H), 4.04 (s, 3H), 4.15 (s, 3H), 6.82 (s,

1H), 7.29-7.32 (m, 2H), 7.35-7.40 (m, 2H), 7.58 (ddd, J = 8.4, 7.3, 1.2 Hz, 1H), 7.71 (d, J = 7.9

Hz, 1H), 7.99 (dd, J = 8.0, 0.9 Hz, 1H), 8.54 (d, J = 7.1 Hz, 1H), 8.71 (d, J = 9.6 Hz, 1H); 13C

NMR (75 MHz, DMSO-d6 at 335 °K) δ 34.11, 42.94, 56.80, 87.96, 99.87, 107.75, 113.02,

117.69, 119.03, 123.10, 124.09, 124.63, 127.97, 128.46, 140.76, 141.03, 145.22, 149.15, 159.74,

+ 163.52; HRMS (ESI) m/z M calcd for C20H19ON2S, 335.12126; found, 335.12093.

8-Methoxy-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1l): Red crystals; 70 mg, yield 24%, starting from 2.6j (200 mg, 0.63 mmol) mp 227-230 °C;

1 H NMR (300 MHz, DMSO-d6) δ 3.98 (s, 3H), 4.01 (s, 3H), 4.36 (s, 3H), 6.79 (s, 1H), 7.33 (d, J

= 7.1 Hz, 1H), 7.40 (ddd, J = 8.0, 7.4, 1.0 Hz, 1H), 7.54-7.58 (m, 1H, overlapped), 7.60 (ddd, J

= 8.5, 7.4, 1.2 Hz, 1H, overlapped), 7.67 (t, J = 8.2 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 8.02 (dd,

122

J = 7.9, 0.8 Hz, 1H), 8.28 (dd, J = 9.0, 1.1 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H); 13C NMR (75

MHz, DMSO-d6) δ 34.21, 49.46, 57.48, 88.60, 108.73, 113.32, 115.52, 117.59, 123.30, 124.20,

124.83, 126.80, 127.82, 128.58, 130.47, 140.96, 147.49, 148.73, 151.75, 160.21; HRMS (ESI)

+ m/z M calcd for C20H19ON2S, 335.12126; found, 335.12091.

5-Chloro-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1m): Red powder; 48 mg, yield 30% starting from 2.12d (110 mg, 0.34 mmol) mp > 300 °C;

1 H NMR (300 MHz, DMSO-d6) δ 3.98 (s, 3H), 4.07 (s, 3H), 7.28 (d, J = 7.2 Hz, 1H), 7.48 (td, J

= 7.6, 1.0 Hz, 1H), 7.61 (s, 1H), 7.66 (ddd, J = 8.5, 7.4, 1.2 Hz, 1H), 7.83-7.91 (m, 3H), 7.96 (dd,

J = 7.6, 2.5 Hz, 1H), 8.12 (dd, J = 7.9, 0.9 Hz, 1H), 8.44 (d, J = 7.2 Hz, 1H); 13C NMR (75

MHz, DMSO-d6) δ 34.62, 43.20, 96.74, 108.85, 114.05, 117.81, 121.50, 123.60, 124.92, 125.59,

128.92, 130.60, 131.80, 132.73, 140.87, 141.32, 144.17, 147.51, 160.94; HRMS (ESI) m/z M+ calcd for C19H16N2SCl, 339.07172 and 341.06877; found, 339.07165 and 341.06841.

6-Chloro-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1n): Red powder; 120 mg, yield 69% starting from 2.12a (120 mg, 0.37 mmol); mp 273-275

1 °C; H NMR (300 MHz, DMSO-d6) δ 4.05 (s, 3H), 4.14 (s, 3H), 6.89 (s, 1H), 7.35 (d, J = 7.2

Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.98-8.06 (m,

13 3H), 8.53 (d, J = 7.2 Hz, 1H), 8.84 (d, J = 2.0 Hz, 1H); C NMR (75 MHz, DMSO-d6 at 310 K)

δ 34.59, 42.87, 89.04, 108.56, 113.62, 120.87, 123.32, 124.44, 124.95, 125.17, 125.67, 128.71,

132.34, 133.53, 137.43, 140.96, 145.44, 148.14, 161.07; HRMS (ESI) m/z M+ calcd for

C19H16N2SCl, 339.07172 and 341.06877; found, 339.07150 and 341.06826.

123

7-Chloro-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1o): Dark red powder; 138 mg, yield 79% starting from 2.12b (120 mg, 0.37 mmol); mp 280-

1 282 °C; H NMR (300 MHz, DMSO-d6 at 310k) δ 4.03 (s, 3H), 4.12 (s, 3H), 6.90 (s, 1H), 7.31

(d, J = 7.2 Hz, 1H), 7.45 (td, J = 7.6, 0.8 Hz, 1H), 7.63 (ddd, J = 8.4, 7.4, 1.1 Hz, 1H), 7.75-7.81

(m, 2H), 8.05 (dd, J = 8.0, 0.8 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H) 8.54 (d, J = 7.29 Hz, 1H), 8.81

13 (d, J = 9.3 Hz, 1H); C NMR (75 MHz, DMSO-d6 at 310 K) δ 34.59, 42.87, 89.04, 108.56,

113.62, 120.87, 120.32, 124.44, 124.95, 125.17, 125.67, 128.71, 132.34, 133.53, 137.43, 140.96,

+ 145.44, 148.14, 161.07; HRMS (ESI) m/z M calcd for C19H16N2SCl, 339.07172 and 341.06877; found, 339.07157 and 341.06845.

8-Chloro-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium triflate

(2.1p): Dark red powder; 79 mg, yield 40% starting from 2.12c (140 mg, 0.40 mmol); mp 288-

1 291 °C; H NMR (300 MHz, DMSO-d6) δ 4.05 (s, 3H), 4.39 (s, 3H), 6.90 (s, 1H), 7.32 (d, J =

7.4 Hz, 1H), 7.46 (td, J = 7.8, 0.7 Hz, 1H), 7.62-7.68 (m, 2H), 7.84 (d, J = 8.3 Hz, 1H), 8.04-

13 8.10 (m, 2H), 8.43 (d, J = 7.3 Hz, 1H), 8.73 (d, J = 7.7 Hz, 1H); C NMR (75 MHz, DMSO-d6)

δ 34.61, 48.69, 90.03, 108.35, 113.94, 121.14 (q, J = 324 Hz), 123.27, 123.48, 124.70, 125.46,

125.76, 127.26, 127.53, 128.82, 136.71, 137.05, 140.89, 148.42, 148.82, 161.73; HRMS (ESI)

+ m/z M calcd for C19H16N2SCl, 339.07172 and 341.06877; found, 339.07162 and 341.06842.

5-Phenyl-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1q): Red powder; 89 mg, yield 57% starting from 2.16a (110 mg, 0.30 mmol); mp 240-243

1 °C; H NMR (300 MHz, DMSO-d6) δ 2.91 (s, 3H), 4.21 (s, 3H), 5.77 (s, 1H), 7.28 (d, J = 7.2 Hz,

1H), 7.36 (ddd, J = 8.0, 7.2, 1.0 Hz, 1H), 7.44-7.64 (m, 8H), 7.97-8.02 (m, 2H), 8.07 (dd, J =

124

13 1.3, 8.7 Hz, 1H), 8.61 (d, J = 7.2 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 32.77, 43.28, 98.17,

109.19, 113.30, 117.91, 122.16, 123.37, 124.32, 124.97, 128.45, 128.60, 129.36, 129.69, 131.37,

132.41, 140.16, 140.58, 141.66, 142.77, 144.51, 149.03, 158.38; HRMS (ESI) m/z M+ calcd for

C25H21N2S, 381.14200; found 381.14155.

6-Phenyl-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1r): Red powder; 50 mg, yield 32% starting from 2.16b (110 mg, 0.30 mmol); mp 270-273

1 °C; H NMR (300 MHz, DMSO-d6) δ 4.05 (s, 3H), 4.20 (s, 3H), 7.04 (s, 1H), 7.40-7.45 (m, 2H),

7.47-7.51 (m, 1H), 7.56-7.64 (m, 3H), 7.78 (d, J = 8.0 1H), 7.93-7.96 (m, 2H), 8.04 (dd, J = 0.7,

7.9 Hz, 1H), 8.13 (d, J = 9.0 Hz, 1H), 8.31 (dd, J = 9.0, 1.7 Hz, 1H), 8.58 (d, J = 7.3 Hz, 1H),

13 8.81 (d, J = 1.8 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 34.35, 42.90, 88.90, 108.54, 113.42,

119.49, 122.93, 123.34, 124.26, 124.81, 124.94, 128.13, 128.62, 128.77, 129.60, 132.60, 137.93,

+ 138.91, 139.13, 140.99, 145.09, 149.02, 160.34; HRMS (ESI) m/z M calcd for C25H21N2S,

381.14200; found, 381.14149.

7-Phenyl-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide

(2.1s): Red powder; 100 mg, yield 64% starting from 2.16c (110 mg, 0.30 mmol); mp 278-280

1 °C; H NMR (300 MHz, DMSO-d6) δ 4.03 (s, 3H), 4.27 (s, 3H), 6.95 (s, 1H), 7.35 (d, J = 7.2 Hz,

1H), 7.41 (td, J = 7.6, 0.8 Hz, 1H), 7.50-7.64 (m, 4H), 7.78 (d, J = 8.2 Hz, 1H), 7.95-8.01 (m,

2H), 8.03-8.09 (m, 2H), 8.18 (d, J = 1.6 Hz, 1H), 8.60 (d, J = 7.2 Hz, 1H), 8.87 (d, J = 9.0 Hz,

13 1H); C NMR (75 MHz, DMSO-d6) δ 34.28, 42.90, 88.46, 108.14, 113.36, 115.94, 123.29,

123.66, 124.30, 124.88, 126.01, 126.78, 128.12, 128.60, 129.57, 129.69, 138.55, 139.00, 140.94,

125

+ 144.92, 145.71, 148.68, 160.23; HRMS (ESI) m/z M calcd for C25H21N2S, 381.14200; found,

381.14163.

8-Phenyl-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidne)methyl)quinolin-1-ium iodide

(2.1t): Red powder; 102 mg, yield 66% starting from 2.16d (110 mg, 0.30 mmol); mp 280-

1 281°C; H NMR (300 MHz, DMSO-d6) δ 3.44 (s, 3H), 4.06 (s, 3H), 6.98 (s, 1H), 7.41-7.57 (m,

7H), 7.63 (ddd, J = 8.4, 7.3, 1.1 Hz, 1H), 7.73-7.83 (m, 3H), 8.07 (dd, J = 7.9, 0.8 Hz, 1H), 8.42

13 (d, J = 7.4 Hz, 1H), 8.82 (dd, J = 7.2, 3.0 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 34.44,

48.45, 89.27, 108.60, 113.60, 123.40, 124.44, 125.11, 125.61, 126.19, 126.42, 128.63, 128.71,

129.02, 129.69, 133.80, 137.66, 137.72, 141.00, 141.33, 147.92, 149.38, 160.86; HRMS (ESI)

+ m/z M calcd for C25H21N2S, 381.14200; found, 381.14154.

126

Synthesis of 1-methyl-4-((3-methylthiazol-2(3H)-ylidene)methyl)-6-phenylquinolin-1-ium iodide (18u)

Compound 2.1u was synthesized following a previously published procedure [150, 151]. To a suspension of 2.6a (0.24 g, 0.84 mmol) and 2.23 [180] (0.24 g, 0.88 mmol) in DCM (15 mL) was added triethylamine (0.3 mL) dropwise and left stirring at rt for 48 h. After completion of the reaction the precipitate was filtered, washed with DCM and the crude product was purified by column chromatography (DCM/MeOH: 9:1) followed by crystallization from DMF to yield 2.1u

1 as dark red crystals; 0.11 g, yield 32%; mp 294-295 °C; H NMR (300 MHz, DMSO-d6) δ 3.94

(s, 3H), 4.02 (s, 3H), 6.69 (s, 1H), 6.92 (d, J = 7.3 Hz, 1H), 7.48 (dd, J = 4.0, 0.9 Hz, 1H), 7.65

(ddd, J = 8.2, 4.7, 3.7 Hz, 1H), 7.85 (d, J = 4.1 Hz, 1H), 7.89-7.91 (m, 2H), 8.29 (d, J = 7.3 Hz,

13 1H), 8.62 (d, J = 8.6 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 38.06, 42.03, 87.58, 105.64,

109.91, 118.15, 123.68, 125.48, 126.48, 132.94, 134.94, 138.44, 144.25, 146.03, 161.80; HRMS

+ (ESI) m/z M calcd for C15H15N2S, 255.09505; found, 255.09469.

127

1-Methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium iodide (2.1v)

Compound 2.1v was prepared following a previously published procedure [150, 151].Yellow powder; 93 mg; yield 27% (starting from 0.21 g of 2.25a, 0.89 mmol and 0.29 g of 2.18a, 0.92

1 mmol); mp 279-281 °C; H NMR (300 MHz, DMSO-d6) δ 3.72 (s, 3H), 3.98 (s, 3H), 6.24 (s,

1H), 7.29 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.37-7.42 (m, 2H), 7.51 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H),

7.58 (dd, J = 8.3, 0.7 Hz, 1H), 7.90 (dd, J =8.0, 0.7 Hz, 1H), 8.27-8.31 (m, 2H); 13C NMR (75

MHz, DMSO-d6) δ 33.31, 45.39, 89.92, 112.43, 118.79, 123.01, 123.72, 123.91, 128.24, 140.98,

+ 142.67, 150.65, 157.36; HRMS (ESI) m/z M calcd for C15H15N2S, 255.09505; found,

255.09485.

128

1-methyl-2-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium iodide (2.1w)

Compound 2.1w was prepared following previously published procedure [150, 151].Orange powder; 170 mg; yield 50% (starting from 0.21 g of 2.25b, 0.89 mmol and 0.29 g of 2.18a, 0.92

1 mmol); mp 287-289 °C; H NMR (300 MHz, DMSO-d6) δ 3.78 (s, 3H), 4.07 (s, 3H), 5.83 (s,

1H), 7.24 (td, J = 6.8, 1.2 Hz, 1H), 7.31 (td, J = 7.6, 1.0 Hz, 1H), 7.52 (ddd, J = 8.3, 7.3, 1.1 Hz,

1H), 7.61 (d, J = 8.0 Hz, 1H), 7.88-7.91 (m, 2H), 8.16 (brt, J = 7.9 Hz, 1H), 8.52 (brd, J = 6.0

13 Hz, 1H); C NMR (75 MHz, DMSO-d6) δ 33.55, 42.77, 82.66, 112.41, 117.27, 120.85, 122.93,

123.06, 124.15, 128.23, 141.26, 142.29, 145.12, 152.02, 158.06; HRMS (ESI) m/z M+ calcd for

C15H15N2S, 255.09505; found, 255.09478.

129

4.2 Arylimidamide azole hybrids.

Synthesis of 4-nitrophenoxy alkyl bromides (3.9a-i).

To a solution of 4-nitrophenol (2.88-14.37 mmol) in dry acetonitrile (20 mL) was added 2 equivalents of K2CO3 (5.76-28.74 mmol) and 5 equivalents of dibromoalakne (14.40- 71.80 mmol). The mixture was stirred at 80 °C overnight according to a previously published procedure [165]. After completion of the reaction, the suspension was filtered and the filtrate was concentrated under reduced pressure. The crude product was subjected to silica gel chromatography employing ethyl acetate/hexanes 1:10 as the eluent to afford the product in 64-

92% yield. For compounds 3.9a-g, the 1H NMR spectra obtained matched data from the literature [165, 181].

1-(2-Bromoethoxy)-4-nitrobenzene (3.9a) [165]. White powder, 2.2 g, yield 62% starting from

2.0 g 4-nitrophenol (14.37 mmol).

130

1-(3-Bromopropoxy)-4-nitrobenzene (3.9b) [165]. White powder, 3.0 g, yield 80% starting from

2.0 g 4-nitrophenol (14.37 mmol).

1-(4-Bromobutoxy)-4-nitrobenzene (3.9c) [165]. White powder, 2.5 g, yield 63% starting from

2.0 g 4-nitrophenol (14.37 mmol).

1-(5-Bromopentoxy)-4-nitrobenzene (3.9d) [181]. White powder, 3.2 g, yield 77% starting from

2.0 g 4-nitrophenol (14.37 mmol).

1-(6-Bromohexoxy)-4-nitrobenzene (3.9e) [181]. Yellow oil, 3.30 g, yield 76% starting from 2.0 g 4-nitrophenol (14.37 mmol).

1-((8-Bromooctyl)oxy)-4-nitrobenzene (3.9f) [181]. White powder, 1.10 g, yield 92% starting from 0.5 g 4-nitrophenol (3.60 mmol).

1-(10-Bromodecyoxy)-4-nitrobenzene (3.9g) [181]. White powder, 1.0 g, yield 78%, starting from 0.5 g 4-nitrophenol (3.60 mmol).

1-((8-Bromooctyl)oxy)-2-chloro-4-nitrobenzene (3.9h). Yellow oil, 0.98 g, yield 93% starting

1 from 0.5 g 2-chloro-4-nitrophenol (2.88 mmol). H NMR (300 MHz, CDCl3) δ 1.35-1.57 (m,

8H), 1.83-1.95 (m, 4H), 3.42 (t, J = 6.8 Hz, 2H), 4.29 (t, J = 6.4 Hz, 2H), 6.98 (d, J = 9.2 Hz,

13 1H), 8.15 (dd, J = 9.2, 2.7 Hz, 1H), 8.29 (d, J = 2.7 Hz, 1H); C NMR (75 MHz, CDCl3) δ

25.75, 28.01, 28.59, 28.74, 29.02, 32.72, 33.90, 69.88, 111.69, 123.42, 123.96, 126.01, 140.99,

159.73.

131

4-((8-Bromooctyl)oxy)-3-chloro-1-nitrobenzene (3.9i). Yellow oil, 0.97 g, yield 86% starting

1 from 0.5 g 3-chloro-4-nitrophenol (2.88 mmol). H NMR (300 MHz, CDCl3) δ 1.36-1.57 (m,

8H), 1.78-1.93 (m, 4H), 3.43 (t, J = 6.8 Hz, 2H), 4.04 (t, J = 6.4 Hz, 2H), 6.87 (dd, J = 9.1, 2.6

13 Hz, 1H), 7.02 (d, J = 2.6 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H); C NMR (75 MHz, CDCl3) δ 25.74,

28.02, 28.60, 28.81, 29.04, 32.71, 33.89, 69.11, 113.38, 117.23, 128.02, 129.68, 140.50, 162.55.

Synthesis of 4-nitrophenoxy alkyl azoles (3.10a-m).

To a solution of 3.6a-i (1.40-5.5 mmol) in dry acetonitrile (20 mL) was added 2 equivalents

K2CO3 (2.80- 11.00 mmol) and 2 equivalents of imidazole or triazole (2.80- 11.00 mmol) and the mixture was stirred at 80 °C overnight. After the reaction was completed, the suspension was filtered and the filtrate was concentrated under reduced pressure. The crude product was

132 subjected to silica gel chromatography employing DCM/MeOH 20:1 as the eluent to afford the product in 51-86% yield. For compounds 3.10a-e, the 1H NMR spectra obtained matched data from the literature [182-184].

1-(2-(4-Nitrophenoxy)ethyl)-1H-imidazole (3.10a) [182]. White powder, 0.72 g, yield 76% starting from 1.00 g 3.9a (4.06 mmol).

1-(2-(4-Nitrophenoxy)ethyl)-1H-1,2,4-triazole (3.10b) [183]. White powder, 0.86 g, yield 75% starting from 1.20 g 3.9a (4.87 mmol).

1-(3-(4-Nitrophenoxy)propyl)-1H-imidazole (3.10c) [184]. White powder, 1.0 g, yield 78% starting from 1.35 g 3.9b (5.19 mmol).

1-(4-(4-Nitrophenoxy)butyl)-1H-imidazole (3.10d) [184]. White powder, 0.73 g, yield 51% starting from 1.5 g 3.9c (5.47 mmol).

1-(4-(4-Nitrophenoxy)butyl)-1H-1,2,4-triazole (3.10e) [184]. White powder, 1.10 g, yield 76% starting from 1.5 g 3.9c (5.47 mmol).

1-(5-(4-Nitrophenoxy)pentyl)-1H-imidazole (3.10f). White powder, 0.96 g, yield 67% starting

1 from 1.5 g 3.9d (5.20 mmol); H NMR (300 MHz, CDCl3) δ 1.45-1.55 (m, 2H), 1.81-1.93 (m,

4H), 3.97-4.06 (m, 4H), 6.90-6.95 (m, 3H), 7.07 (s, 1H), 7.48 (s, 1H), 8.16-8.22 (m, 2H); 13C

NMR (75 MHz, CDCl3) δ 23.14, 28.48, 30.78, 46.80, 68.28, 114.36, 118.71, 125.90, 129.59,

137.06, 141.49, 163.91.

133

1-(6-(4-Nitrophenoxy)hexyl)-1H-imidazole (3.10g). White powder, 1.10 g, yield 72% starting

1 from 1.6 g 3.9e (5.29 mmol); H NMR (300 MHz, CDCl3) δ 1.33-1.43 (m, 2H), 1.47-1.57 (m,

2H), 1.77-1.88 (m, 4H), 3.96 (t, J = 7.0 Hz, 2H), 4.03 (t, J = 6.3 Hz, 2H), 6.90-6.96 (m, 3H), 7.06

13 (s, 1H), 7.47 (s, 1H), 8.17-8.22 (m, 2H); C NMR (75 MHz, CDCl3) δ 25.52, 26.27, 28.80,

30.96, 46.87, 68.48, 114.36, 118.71, 125.90, 129.51, 137.05, 141.43, 164.04.

1-(8-(4-Nitrophenoxy)octyl)-1H-imidazole (3.10h). Off-white powder, 0.41 g, yield 86% starting

1 from 0.5 g 3.9f (1.51 mmol); H NMR (300 MHz, CDCl3) δ 1.26-1.51 (m, 8H), 1.74-1.86 (m,

4H), 3.94 (t, J = 7.1 Hz, 2H), 4.04 (t, J = 6.5 Hz, 2H), 6.90-6.97 (m, 3H), 7.06 (s, 1H), 7.47 (s,

13 1H), 8.17-8.22 (m, 2H); C NMR (75 MHz, CDCl3) δ 25.80, 26.45, 28.90, 28.96, 29.10, 31.02,

46.96, 68.73, 114.37, 118.71, 125.89, 129.46, 137.07, 141.37, 164.16.

1-(8-(4-Nitrophenoxy)octyl)-1H-1,2,4-triazole (3.10i). White powder, 0.41 g, yield 70% starting

1 from 0.6 g 3.9f (1.81 mmol); H NMR (400 MHz, CDCl3) δ 1.27-1.50 (m, 8H), 1.79-1.86 (m,

2H), 1.88-1.95 (m, 2H), 4.05 (t, J = 6.4 Hz, 2H), 4.18 (t, J = 7.1 Hz, 2H), 6.93-6.97 (m, 2H), 7.95

13 (s, 1H), 8.06 (s, 1H), 8.18-8.22 (m, 2H); C NMR (100 MHz, CDCl3) δ 25.80, 26.37, 28.91

(overlapped 2Cs), 29.06, 29.74, 49.66, 68.74, 114.38, 125.91, 141.37, 142.79, 151.93, 164.17.

1-(10-(4-Nitrophenoxy)decyl)-1H-imidazole (3.10j). Off-white powder, 0.32 g, yield 65%,

1 starting from 0.5 g 3.9g (1.40 mmol); H NMR (300 MHz, CDCl3) δ 1.26-1.51 (m, 12H), 1.75-

1.87 (m, 4H), 3.93 (t, J = 7.1 Hz, 2H), 4.05 (t, J = 6.5 Hz, 2H), 6.91-6.97 (m, 3H), 7.06 (s, 1H),

13 7.46 (s, 1H), 8.17-8.22 (m, 2H); C NMR (75 MHz, CDCl3) δ 25.86, 26.51, 28.94, 29.02, 29.22,

29.31, 29.35, 31.03, 46.99, 68.84, 114.38, 118.72, 125.88, 129.39, 137.06, 141.34, 164.21.

134

Synthesis of 1-(8-(4-nitrophenoxy)octyl)-1H-pyrrole (3.10k). 3.10k was synthesized according to a previously published procedure [166]. To a solution of pyrrole (0.21 g, 3.13 mmol) in DMF

(5 mL) was added 1.1 equivalent of potassium hydroxide (0.20 g, 3.56 mmol) and the suspension was stirred at rt for 15 min. 1.1 equivalent of 3.9f (1.2 g, 3.56 mmol) was added and the mixture was stirred at 80 °C for 16h. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate, and dried over sodium sulfate. The organic layer was evaporated under reduced pressure and purified by column chromatography using hexanes/DCM

1 (5:1) as the eluent to yield the product as a yellow oil, 0.35 g, 35. H NMR (300 MHz, CDCl3) δ

1.28-1.50 (m, 8H), 1.74-1.87 (m, 4H), 3.89 (t, J = 7.1 Hz, 2H), 4.05 (t, J = 6.5 Hz, 2H), 6.15 (t, J

= 2.1 Hz, 2H), 6.66 (t, J = 2.1 Hz, 2H), 6.92-6.98 (m, 2H), 8.18-8.24 (m, 2H); 13C NMR (75

MHz, CDCl3) δ 25.82, 26.67, 28.92, 29.09, 29.13, 31.51, 49.57, 68.80, 107.80, 114.39, 120.44,

125.90, 141.36, 164.21.

1-(8-(2-Chloro-4-nitrophenoxy)octyl)-1H-imidazole (3.10l). Yellow oil, 0.41 g, yield 60%,

1 starting from 0.71 g 3.9h (1.94 mmol); H NMR (400 MHz, CDCl3) δ 1.32-1.53 (m, 8H), 1.76-

1.91 (m, 4H), 3.94 (t, J = 7.0 Hz, 2H), 4.13 (t, J = 6.4 Hz, 2H), 6.91 (s, 1H), 6.97 (d, J = 9.1 Hz,

1H), 7.06 (s, 1H), 7.46 (s, 1H), 8.14 (dd, J = 9.1, 2.7 Hz, 1H), 8.29 (d, J = 2.7 Hz, 1H); 13C NMR

(100 MHz, CDCl3) δ 25.75, 26.44, 28.72, 28.94, 29.02, 31.02, 46.99, 69.82, 111.70, 118.73,

123.37, 123.98, 125.99, 129.41, 137.06, 140.99, 159.70.

1-(8-(3-Chloro-4-nitrophenoxy)octyl)-1H-imidazole (3.10m). Synthesis of 3.10m followed the general synthesis procedure except for the use of 1.2 equivalents of imidazole (115 mg, 1.69 mmol) and 1.2 equivalents of potassium carbonate (231 mg, 1.66 mmol) with gentle reflux

135 overnight. Yellow oil, 0.24 g, yield 49% starting from 0.51 g 3.9i (1.39 mmol). 1H NMR (400

MHz, CDCl3) δ 1.26-1.49 (m, 8H), 1.73-1.86 (m, 4H), 3.94 (t, J = 7.1 Hz, 2H), 4.02 (t, J = 6.5

Hz, 2H), 6.85 (dd, J = 9.2, 2.6 Hz, 1H), 6.91 (s, 1H), 7.00 (d, J = 2.6 Hz, 1H), 7.06 (s, 1H), 7.46

13 (s, 1H), 8.00 (d, J = 9.2 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.74, 26.45, 28.79, 28.96,

29.06, 31.02, 46.98, 69.06, 113.37, 117.23, 118.74, 128.03, 129.41, 129.66, 137.06, 140.49,

162.53.

Synthesis of 4-aminophenoxy alkyl azoles (3.11a-m).

To a solution of 3.10a-m (0.60-1.70 mmol) in ethyl acetate (20 mL) was added 5 equivalents of tin(II) chloride dihydrate (3.0-8.5 mmol) and the mixture was refluxed for 8-12 h. After the reaction was comple, the suspension was rendered alkaline with 1N NaOH solution (pH = 11)

136 and extracted with ethyl acetate (30 mL × 3). The organic layer was dried over sodium sulfate and filtered. The filtrate was evaporated under reduced pressure yielding the product in 81-99% yield which was taken directly to the next step without further purification.

4-(2-(1H-imidazol-1-yl)ethoxy)aniline (3.11a). Yellow powder, 0.10 g, yield 82% starting from

1 0.14 g 3.10a (0.60 mmol). H NMR (300 MHz, DMSO-d6) δ 4.07 (t, J = 5.2 Hz, 2H), 4.27 (t, J =

5.2 Hz, 2H), 4.61 (brs, 2H), 6.45-6.51 (m, 2H), 6.60-6.65 (m, 2H), 6.88 (s, 1H), 7.21 (s, 1H),

7.64 (s, 1H).

4-(2-(1H-1,2,4-triazol-1-yl)ethoxy)aniline (3.11b). Yellow powder, 0.28 g, yield 81% starting

1 from 0.40 g 3.10b (1.70 mmol). H NMR (300 MHz, CDCl3) δ 3.50 (brs, 2H), 4.26 (t, J = 5.1

Hz, 2H), 4.52 (t, J = 5.1 Hz, 2H), 6.59-6.64 (m, 2H), 6.66-6.76 (m, 2H), 7.96 (s, 1H), 8.22 (s,

1H).

4-(3-(1H-imidazol-1-yl)propoxy)aniline (3.11c). Brown oil, 0.26 g, yield 99% starting from 0.30

1 g 3.10c (1.21 mmol). H NMR (300 MHz, CDCl3) δ 2.15-2.23 (m, 2H), 3.14 (brs, 2H), 3.85 (t, J

= 5.6 Hz, 2H), 4.18 (t, J = 6.9 Hz, 2H), 6.62-6.67 (m, 2H), 6.71-6.75 (m, 2H), 6.93 (s, 1H), 7.07

(s, 1H), 7.49 (s, 1H).

4-(4-(1H-imidazol-1-yl)butoxy)aniline (3.11d). Buff powder, 0.25 g, yield 95% starting from 0.3

1 g 3.10d (1.14 mmol); H NMR (300 MHz, DMSO-d6) δ 1.70-1.79 (m, 2H), 1.93-2.00 (m, 2H),

3.28 (brs, 2H), 3.90 (t, J = 6.0 Hz, 2H), 4.02 (t, J = 7.1 Hz, 2H), 6.62-6.66 (m, 2H), 6.71-6.75

(m, 2H), 6.93 (s, 1H), 7.07 (s, 1H), 7.49 (s, 1H).

137

4-(4-(1H-1,2,4-triazol-1-yl)butoxy)aniline (3.11e). Buff powder, 0.32 g, yield 91% starting from

1 0.40 g 3.10e (1.52 mmol); H NMR (300 MHz, CDCl3) δ 1.71-1.80 (m, 2H), 2.05-2.14 (m, 2H),

3.45 (brs, 2H), 3.92 (t, J = 6.0 Hz, 2H), 4.26 (t, J = 7.1 Hz, 2H), 6.61-6.66 (m, 2H), 6.70-6.75

(m, 2H), 7.95 (s, 1H), 8.08 (s, 1H).

4-((5-(1H-imidazole-1-yl)pentyl)oxy)aniline (3.11f). Brown oil, 0.29 g, yield 99% starting from

1 0.33 g 3.10f (1.19 mmol); H NMR (300 MHz, CDCl3) δ 1.42-1.52 (m, 2H), 1.72-1.90 (m, 4H),

3.23 (brs, 2H), 3.88 (t, J = 6.2 Hz, 2H), 3.96 (t, J = 7.1 Hz, 2H), 6.61-6.66 (m, 2H), 6.70-6.75

(m, 2H), 6.91 (s, 1H), 7.06 (s, 1H), 7.48 (s, 1H).

4-((6-(1H-imidazole-1-yl)hexyl)oxy)aniline (3.11g). Brown oil, 0.30 g, yield 92% starting from

1 0.35 g 3.10g (1.20 mmol); H NMR (300 MHz, CDCl3) δ 1.33-1.42 (m, 2H), 1.45-1.55 (m, 2H),

1.69-1.86 (m, 4H), 3.17 (brs, 2H), 3.88 (t, J = 6.3 Hz, 2H), 3.94 (t, J = 7.1 Hz, 2H), 6.60-6.67 (m,

2H), 6.71-6.76 (m, 2H), 6.91 (s, 1H), 7.07 (s, 1H), 7.47 (s, 1H).

4-((8-(1H-imidazole-1-yl)octyl)oxy)aniline (3.11h). Orange powder, 0.23 g, yield 85%, starting

1 from 0.30 g 3.10h (0.95 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.50 (m, 8H), 1.69-1.84 (m,

4H), 3.88 (t, J = 6.5 Hz, 2H), 3.94 (t, J = 7.2 Hz, 2H), 5.25 (brs, 2H), 6.63-6.68 (m, 2H), 6.72-

6.77 (m, 2H), 6.91 (t, J = 1.2 Hz, 1H), 7.09 (s, 1H), 7.61 (s, 1H).

4-((8-(1H-1,2,4-triazol-1-yl)octyl)oxy)aniline (3.11i). Buff powder, 0.21 g, yield 93%, starting

1 from 0.25g 3.10i (0.78 mmol); H NMR (400 MHz, CDCl3) δ 1.27-1.45 (m, 8H), 1.70-1.77 (m,

2H), 1.86-1.93 (m, 2H), 3.44 (brs, 2H), 3.87 (t, J = 6.4 Hz, 2H), 4.16 (t, J = 7.1 Hz, 2H), 6.62-

6.66 (m, 2H), 6.72-6.77 (m, 2H), 7.95 (s, 1H), 8.05 (s, 1H).

138

4-((10-(1H-imidazole-1-yl)decyl)oxy)aniline (3.11j). Buff powder, 0.20 g, yield 88%, starting

1 from 0.25 g 3.10j (0.72 mmol); H NMR (300 MHz, CDCl3) δ 1.30-1.47 (m, 12H), 1.69-1.80 (m,

4H), 3.36 (brs, 2H), 3.86-3.95 (m, 4H), 6.61-6.67 (m, 2H), 6.72-6.78 (m, 2H), 6.91 (t, J = 1.2 Hz,

1H), 7.07 (s, 1H), 7.47 (s, 1H).

Synthesis of 4-((8-(1H-pyrrol-1-yl)octyl)oxy)aniline (3.11k). Brown oil, 0.26 g, yield 96%,

1 starting from 0.30 g 3.10k (0.95 mmol); H NMR (300 MHz, CDCl3) δ 1.26-1.48 (m, 8H), 1.70-

1.85 (m, 4H), 3.37 (brs, 2H), 3.85-3.91 (m, 4H), 6.15 (t, J = 2.0 Hz, 2H) 6.62-6.69 (m, 4H), 6.74-

6.78 (m, 2H).

4-((8-(1H-imidazole-1-yl)octyl)oxy)-3-chloroaniline (3.11l). yellow oil, 0.17 g, yield 93%,

1 starting from 0.20 g 3.10l (0.57 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.43 (m, 8H), 1.72-

1.84 (m, 4H), 3.35 (brs, 2H), 3.93 (t, J = 6.7 Hz, 4H), 6.54 (dd, J = 8.6, 2.8 Hz, 1H), 6.74 (d, J =

2.8 Hz, 1H, overlapped), 6.77 (d, J = 8.6 Hz, 1H, overlapped), 6.91 (s, 1H), 7.06 (s, 1H), 7.47 (s,

1H).

4-((8-(1H-imidazole-1-yl)octyl)oxy)-2-chloroaniline (3.11m). yellow oil, 0.23 g, yield 97%,

1 starting from 0.26 g 3.10m (0.74 mmol); H NMR (400 MHz, CDCl3) δ 1.25-1.48 (m, 8H), 1.70-

1.83 (m, 4H), 3.76 (brs, 2H), 3.87 (t, J = 6.5 Hz, 2H), 3.94 (t, J = 7.1 Hz, 2H), 6.67-6.73 (m, 2H),

6.85 (d, J = 2.4 Hz, 1H), 6.92 (s, 1H), 7.07 (s, 1H), 7.48 (s, 1H).

139

Synthesis of N-(4-(1H-imidazol-1-yl)alkoxyphenyl) picolinimidamide 3.12a-n

Synthesis of AIA azole hybrids followed previously published procedures [117, 122, 185]. Two equivalents of S-(2-naphthylmethyl)-2-pyridyl thioimidate hydrobromide (0.76-1.38 mmol) were added to one equivalent of a cooled solution of the arylamines 3.8a-m (0.38-0.69 mmol) in dry acetonitrile: ethanol (3:10 mL) in an ice bath for 15 minutes, then this solution was warmed to room temperature. The reaction mixture was stirred at room temperature for 24-48 h. After the disappearance of the starting material, the organic solvent was evaporated under reduced pressure to yield a crude oil product. Dry ether (100 mL) was added to the crude material and the mixture was stirred at room temperature overnight. The precipitate was filtered and washed with

140 dry ether. The solid was dissolved in ethanol (2 ml), then the solution was cooled to 0 oC in an ice bath and 10% NaOH was added until the pH reached approximately 11. The free base was extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with distilled water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure yielding a waxy or solid product which was further purified by column chromatography on triethylamine neutralized silica using DCM:MeOH as an eluent (100:0.5 to 100:3). The product was further crystalized using hexanes/ethyl acetate to yield the final product in 33-70% yield.

N-(4-(2-(1H-imidazol-1-yl)ethoxy)phenyl) picolinimidamide 3.12a. Yellow powder; 63 mg; yield 42% (starting from 0.10 g of 3.11a, 0.49 mmol); mp 141-144 °C; 1H NMR (400 MHz,

DMSO-d6) δ 4.22 (t, J = 5.1 Hz, 2H), 4.35 (t, J = 5.1 Hz, 2H), 6.44 (brs, 2H), 6.82-6.95 (m, 5H),

7.26 (t, J = 1.1 Hz, 1H), 7.54 (ddd, J = 7.4, 4.8, 1.2 Hz, 1H), 7.69 (s, 1H), 7.93 (td, J = 7.7, 1.8

Hz, 1H), 8.29 (d, J = 7.9 Hz, 1H), 8.62 (ddd, J = 4.8, 1.7, 0.9 Hz, 1H); 13C NMR (100 MHz,

DMSO-d6) δ 46.15, 67.89, 115.83, 120.18, 121.63, 122.92, 125.76, 128.74, 137.53, 138.09,

+ 144.08, 148.44, 152.09, 152.33, 153.91; HRMS (ESI) m/z (M +H) calcd for C17H18N5O,

308.15059; found, 308.15057; Anal. Calcd for C17H17N5O: C, 66.43; H, 5.58; N, 22.79. Found:

C, 66.15; H, 5.71; N, 22.49.

N-(4-(2-(1H-1,2,4-triazol-1-yl)ethoxy)phenyl) picolinimidamide 3.12b. Buff powder; 107 mg; yield 59% (starting from 0.12 g of 3.11b, 0.58 mmol); mp 115-118 °C1H NMR (400 MHz,

CDCl3) δ 4.35 (t, J = 5.0 Hz, 2H), 4.58 (t, J = 5.0 Hz, 2H), 5.85 (brs, 2H), 6.86-6.91 (m, 2H),

6.93-6.98 (m, 2H), 7.40 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 7.82 (td, J = 7.7, 1.7 Hz, 1H), 7.98 (s,

1H), 8.25 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.58 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H); 13C NMR (100

141

MHz, CDCl3) δ 49.42, 66.09, 115.79, 121.49, 122.61, 125.12, 136.76, 144.01, 147.85, 151.57,

+ 152.05, 152.95, 153.87; HRMS (ESI) m/z (M +H) calcd for C16H17N6O, 309.14584; found,

309.14569; Anal. Calcd for C16H16N6O: C, 62.32; H, 5.23; N, 27.26. Found: C, 62.39; H, 5.28; N,

27.18.

N-(4-(3-(1H-imidazol-1-yl)propoxy)phenyl) picolinimidamide 3.12c. Yellow powder; 85 mg; yield 38% (starting from 0.15 g of 3.11c, 0.69 mmol); mp 115-118 °C; 1H NMR (700 MHz,

CDCl3) δ 2.13-2.17 (m, 2H), 3.83 (t, J = 5.6 Hz, 2H), 4.13 (t, J = 6.8 Hz, 2H), 5.80 (m, 2H),

6.81-6.84 (m, 2H), 6.86 (t, J = 1.2 Hz, 1H), 6.88-6.90 (m, 2H), 6.99 (s, 1H), 7.32 (ddd, J = 1.1,

4.8, 7.5 Hz, 1H), 7.42 (s, 1H), 7.73 (td, J = 1.7, 7.7 Hz, 1H), 8.33 (d, J = 7.9 Hz, 1H), 8.50 (ddd,

13 J = 0.9, 1.6, 4.8, 1H); C NMR (175 MHz, CDCl3) δ 30.89, 43.48, 64.01, 115.56, 118.97,

121.52, 122.64, 125.13, 129.69, 136.79, 137.42, 143.36, 147.86, 151.60, 153.02, 154.54; HRMS

+ (ESI) m/z (M +H) calcd for C18H20N5O, 322.16624; found, 322.16952; Anal. Calcd for

C18H19N5O: C, 67.27; H, 5.96; N, 21.79. Found: C, 67.42; H, 5.98; N, 27.78.

N-(4-(4-(1H-imidazol-1-yl)butoxy)phenyl) picolinimidamide 3.12d. Buff powder; 132 mg; yield

1 65% (starting from 0.14 g of 3.11d, 0.60 mmol); mp 103-105 °C; H NMR (400 MHz, CDCl3) δ

1.76-1.84 (m, 2H), 1.98-2.07 (m, 2H), 3.99 (t, J = 6.0 Hz, 2H), 4.05 (t, J = 7.0 Hz, 2H), 5.86 (brs,

2H), 6.89-7.00 (m, 5H), 7.09 (t, J = 0.9 Hz, 1H), 7.40 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H), 7.51 (s,

1H), 7.82 (td, J = 7.7, 1.7 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.58 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H);

13 C NMR (100 MHz, CDCl3) δ 26.36, 28.11, 46.78, 67.40, 115.52, 118.76, 121.49, 122.54,

125.09, 129.55, 136.76, 137.11, 143.21, 147.84, 151.66, 152.94, 154.77; HRMS (ESI) m/z (M

142

+ +H) calcd for C19H22N5O, 336.18189; found, 336.18155; Anal. Calcd for C19H21N5O: C, 68.04;

H, 6.31; N, 20.88. Found: C, 68.20; H, 6.35; N, 20.77.

N-(4-(4-(1H-1,2,4-triazol-1-yl)butoxy)phenyl) picolinimidamide 3.12e. Buff powder; 141 mg; yield 70% (starting from 0.14 g of 3.11e, 0.60 mmol); mp 109-111 °C; 1H NMR (400 MHz,

CDCl3) δ 1.78-1.84 (m, 2H), 2.10-2.18 (m, 2H), 4.01 (t, J = 6.0 Hz, 2H), 4.29 (t, J = 7.0 Hz, 2H),

5.88 (brs, 2H), 6.90-6.93 (m, 2H), 6.96-6.98 (m, 2H), 7.40 (ddd, J = 7.7, 4.9, 1.2 Hz, 1H), 7.82

(td, J = 7.6, 1.7 Hz, 1H), 7.96 (s, 1H), 8.10 (s, 1H), 8.42 (d, J = 7.9 Hz, 1H), 8.58 (ddd, J = 4.8,

13 1.7, 0.9 Hz, 1H); C NMR (100 MHz, CDCl3) δ 26.21, 26.92, 49.39, 67.36, 115.51, 121.49,

122.55, 125.08, 136.75, 142.94, 143.96, 147.84, 151.67, 152.01, 152.96, 154.75; HRMS (ESI)

+ m/z (M +H) calcd for C18H21N6O, 337.17714; found, 337.17712; Anal. Calcd forC18H21N6O: C,

64.27; H, 5.99; N, 24.98. Found: C, 64.43; H, 6.00; N, 24.93.

N-(4-((5-(1H-imidazol-1-yl)pentyl)oxy)phenyl) picolinimidamide 3.12f. Light brown powder; 70 mg; yield 33% (starting from 0.15 g of 3.11f, 0.61 mmol); mp 128-131 °C; 1H NMR (400 MHz,

CDCl3) δ 1.48-1.56 (m, 2H), 1.78-1.93 (m,4H), 3.95-4.03 (m, 4H), 5.85 (brs, 2H), 6.89-6.95 (m,

3H), 6.96-7.00 (m, 2H), 7.08 (s, 1H), 7.40 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.49 (s, 1H), 7.82 (td,

J = 7.7, 1.7 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.58 (ddd, J = 4.8, 1.4, 0.8 Hz, 1H); 13C NMR

(100 MHz, CDCl3) δ 23.31, 28.80, 30.89, 46.91, 67.70, 115.51, 118.75, 121.49, 122.51, 125.06,

129.51, 136.74, 137.08, 143.06, 147.83, 151.70, 152.94, 154.94 ; HRMS (ESI) m/z (M +H)+ calcd for C20H24N5O, 350.19754; found, 350.19757; Anal. Calcd for C20H23N5O: C, 68.74; H,

6.63; N, 20.04. Found: C, 68.45; H, 6.60; N, 19.90.

143

N-(4-((6-(1H-imidazol-1-yl)hexyl)oxy)phenyl) picolinimidamide 3.12g. Light brown powder; 70 mg; yield 33% (starting from 0.15 g of 3.11g, 0.57 mmol); mp 127-129 °C; 1H NMR (400 MHz,

CDCl3) δ 1.36-1.44 (m, 2H), 1.50-1.58 (m, 2H), 1.76-1.88 (m, 4H), 3.95-3.99 (m, 4H), 5.89 (brs,

2H), 6.91-7.01 (m, 5H), 7.08 (s, 1H), 7.41 (ddd, J = 7.7, 4.9, 1.0 Hz, 1H), 7.48 (s, 1H), 7.83 (td, J

= 7.7, 1.6 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 4.6 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 25.64, 26.32, 29.17, 31.03, 46.92, 67.89, 115.52, 118.74, 121.49, 122.47, 125.06,

129.46, 136.74, 137.08, 142.97, 147.83, 151.72, 152.90, 155.05; HRMS (ESI) m/z (M +H)+ calcd for C21H26N5O, 361.21319; found, 361.21298; Anal. Calcd for C21H25N5O: C, 69.40; H, 6.93; N,

19.27. Found: C, 69.43; H, 6.81; N, 19.13.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)phenyl) picolinimidamide 3.12h. Light brown powder; 85 mg; yield 44% (starting from 0.14 g of 3.11h, 0.49 mmol); mp 89-92 °C; 1H NMR (400 MHz,

CDCl3) δ 1.29-1.40 (m, 8H), 1.76-1.84 (m, 4H), 3.93-3.98 (m, 4H), 5.93 (brs, 2H), 6.92-6.95 (m,

3H), 6.97-6.99 (m, 2H), 7.07 (t, J = 1.0 Hz, 1H), 7.41 (ddd, J = 7.5, 4.9, 1.2 Hz, 1H), 7.47 (s,

1H), 7.83 (td, J = 7.6, 1.7 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.59 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H);

13 C NMR (100 MHz, CDCl3) δ 25.97, 26.49, 29.00, 29.17, 29.30, 31.06, 47.02, 68.17, 115.55,

118.74, 121.53, 122.55, 125.09, 129.42, 136.76, 137.08, 142.56, 147.84, 151.64, 153.05, 155.23;

+ HRMS (ESI) m/z (M +H) calcd for C23H30N5O, 392.24449; found, 392.24451; Anal. Calcd for

C23H29N5O: C, 70.56; H, 7.47; N, 17.89. Found: C, 70.27; H, 7.29; N, 17.70.

N-(4-((8-(1H-1,2,4-triazol-1-yl)octyl)oxy)phenyl) picolinimidamide 3.12i. Light brown powder;

85 mg; yield 39% (starting from 0.16 g of 3.11i, 0.55 mmol); mp 98-100 °C; 1H NMR (700

MHz, CDCl3) δ 1.31-1.51 (m, 6H), 1.45-1.50 (m, 2H), 1.76-1.81 (m, 2H), 1.89-1.94 (m, 2H),

144

3.96 (t, J = 6.4 Hz, 2H), 4.18 (t, J = 7.1 Hz, 2H), 5.91 (brs, 2H), 6.91-6.94 (m, 2H), 6.96-6.99 (m,

2H), 7.40 (ddd, J = 7.3, 4.8, 0.9 Hz, 1H), 7.82 (td, J = 7.7, 1.7 Hz, 1H), 7.95 (s, 1H), 8.06 (s,

13 1H), 8.43 (d, J = 7.9 Hz, 1H), 8.58 (d, J = 4.7 Hz, 1H) ; C NMR (100 MHz, CDCl3) δ 25.96,

26.39, 28.94, 29.15, 29.31, 29.77, 49.71, 68.16, 115.54, 121.54, 122.56, 125.11, 136.78, 142.79,

+ 147.85, 151.61, 151.92, 153.05, 155.24; HRMS (ESI) m/z (M +H) calcd for C22H29N6O,

393.23974; found, 393.23932; Anal. Calcd for C22H28N6O: C, 67.32; H, 7.19; N, 21.41. Found:

C, 67.18; H, 7.16; N, 21.47.

N-(4-((10-(1H-imidazol-1-yl)decyl)oxy)phenyl) picolinimidamide 3.12j. Light brown powder;

61 mg; yield 38% (starting from 0.12 g of 3.11j, 0.38 mmol); mp 104-106 °C; 1H NMR (400

MHz, CDCl3) δ 1.27-1.41 (m, 10H), 1.44-1.50 (m, 2H), 1.76-1.83 (m, 4H), 3.92-3.98 (m, 4H),

5.85 (brs, 2H), 6.92-6.99 (m, 5H), 7.07 (s, 1H), 7.40 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 7.47 (s, 1H),

7.82 (td, J = 7.7, 1.7 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.59 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H); 13C

NMR (100 MHz, CDCl3) δ 26.04, 26.54, 29.04, 29.33 (overlapped 2Cs), 29.36, 29.41, 31.07,

47.03, 68.27, 115.55, 118.74, 121.52, 122.53, 125.07, 129.39, 136.75, 137.07, 142.35, 147.83,

+ 151.65, 153.04, 155.26; HRMS (ESI) m/z (M +H) calcd for C25H34N5O, 420.27579; found,

420.27637; Anal. Calcd for C25H33N5O: C, 71.57; H, 7.93; N, 16.69. Found: C, 71.27; H, 7.66; N,

16.47.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)phenyl)benzimidamide 3.12k. Buff powder; 45 mg; yield

1 22% (starting from 0.15 g of 3.11h, 0.52 mmol); mp 107-109 °C; H NMR (700 MHz, CDCl3) δ

1.30-1.41 (m, 6H), 1.44-1.51 (m, 2H), 1.76-1.82 (m, 4H), 3.92-3.97 (m, 4H), 4.94 (brs, 2H),

6.90-6.95 (m, 5H), 7.06 (d, J = 0.5 Hz, 1H), 7.43-7.51 (m, 4H), 7.87 (brs, 2H); 13C NMR (100

145

MHz, CDCl3) δ 25.98, 26.49, 29.01, 29.18, 29.32, 31.06, 47.02, 68.15, 115.51, 118.76, 122.55,

126.77, 128.53, 129.42, 130.50, 135.97, 137.08, 142.49, 155.13; HRMS (ESI) m/z (M +H)+ calcd for C24H31N4O, 391.24924; found, 391.24905; Anal. Calcd for C24H30N4O: C, 73.81; H, 7.74; N,

14.35. Found: C, 73.53; H, 7.80; N, 14.26.

Synthesis of N-(4-((8-(1H-pyrrol-1-yl)octyl)oxy)phenyl) picolinimidamide 3.12l. The synthesis of 3.12l follows the general synthesis of 3.12a-k with the modification of using 1.5 equivalents of S-(2-naphthylmethyl)-2-pyridyl thioimidate hydrobromide and, after completion of the reaction, no diethyl ether treatment was performed. The compound was purified by column chromatography using hexanes/DCM (1:1.5) on triethylamine neutralized silica. Light brown powder, 58 mg, yield 17% (starting from 0.25 g of 3.11k, 0.87 mmol); mp 93-95 °C; 1H NMR

(400 MHz, CDCl3) δ 1.31-1.49 (m, 8H), 1.76-1.83 (m, 4H), 3.89 (t, J = 7.2 Hz, 2H), 3.96 (t, J =

6.5 Hz, 2H), 5.87 (brs, 2H), 6.16 (t, J = 2.1 Hz, 2H), 6.67 (t, J = 2.1 Hz, 2H), 6.92-6.99 (m, 4H),

7.41 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 7.83 (td, J = 7.7, 1.7 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H), 8.60

13 (d, J = 4.8 Hz, 1H) ; C NMR (100 MHz, CDCl3) δ 26.01, 26.71, 29.15, 29.26, 29.34, 31.55,

49.61, 68.23, 107.77, 115.55, 120.45, 121.50, 122.44, 125.05, 136.74, 142.82, 147.83, 151.74,

+ 152.89, 155.21; HRMS (ESI) m/z (M +H) calcd for C24H31N4O, 391.24924; found, 391.24988;

Anal. Calcd for C24H30N4O: C, 73.81; H, 7.74; N, 14.35. Found: C, 74.03; H, 7.89; N, 14.25.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-chlorophenyl) picolinimidamide 3.12m. Yellow powder; 99 mg; yield 54% (starting from 0.14 g of 3.11l, 0.43 mmol); mp 102-104 °C; 1H NMR

(400 MHz, CDCl3) δ 1.29-1.45 (m, 6H), 1.47-1.57 (m, 2H), 1.76-1.88 (m, 4H), 3.94 (t, J = 7.1

Hz, 2H), 4.03 (t, J = 6.4 Hz, 2H), 5.93 (brs, 2H), 6.89 (dd, J = 8.6, 2.4 Hz, 1H), 6.92 (t, J = 1.1

146

Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 7.07 (s, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.41 (ddd, J = 7.5, 4.8,

1.2 Hz, 1H), 7.47 (s, 1H), 7.83 (td, J = 7.7, 1.7 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H), 8.59 (ddd, J =

13 4.8, 1.7, 0.9 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.86, 26.45, 28.95, 29.07, 29.14, 31.04,

47.02, 69.55, 114.87, 118.74, 120.69, 121.52, 123.49, 123.72, 125.23, 129.41, 136.81, 137.07,

143.47, 147.89, 150.60, 151.34, 153.20; HRMS (ESI) m/z (M +H)+ and ((M+2) +H)+ calcd for

C23H29N5ClO, 426.20551 and 428.20261; found, 426.20490 and 428.20236; Anal. Calcd for

C23H28N5ClO: C, 64.85; H, 6.63; N, 16.44. Found: C, 64.81; H, 6.67; N, 16.32.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)-2-chlorophenyl) picolinimidamide 3.12n. white powder;

71 mg; yield 39% (starting from 0.14 g of 3.11m, 0.43 mmol); mp 74-76 °C; 1H NMR (400

MHz, CDCl3) δ 1.28-1.42 (m, 6H), 1.42-1.51 (m, 2H), 1.76-1.82 (m, 4H), 3.93-3.97 (m, 4H),

5.79 (brs, 2H), 6.84 (dd, J = 8.7, 2.7 Hz, 1H), 6.92 (s, 1H), 6.97 (d, J = 8.7 Hz, 1H), 7.02 (d, J =

2.7 Hz, 1H), 7.08 (s, 1H), 7.42 (ddd, J = 7.5, 4.8, 1.0 Hz, 1H), 7.48 (s, 1H), 7.84 (td, J = 7.8, 1.6

13 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 4.8 Hz, 1H); C NMR (100 MHz, CDCl3) δ

25.90, 26.48, 28.99, 29.13, 29.16, 31.06, 47.01, 68.42, 114.61, 116.02, 118.74, 121.83, 123.27,

125.29, 126.54, 129.43, 136.87, 137.07, 139.60, 147.90, 151.18, 153.22, 155.50; HRMS (ESI)

+ + m/z (M +H) and ((M+2) +H) calcd for C23H29N5ClO, 426.20551 and 428.20261; found,

426.20703 and 428.20465; Anal. Calcd for C23H28N5ClO: C, 64.85; H, 6.63; N, 16.44. Found: C,

64.68; H, 6.58; N, 16.38.

147

Synthesis of 2-(methoxymethoxy)-5-nitrophenol (3.14)

3.14 was synthesized following a previously published procedure [167]. To a solution of 3.13

(1.0 g, 6.44 mmol) in dry DMF (10 mL) was added 1.2 equivalent of potassium carbonate (1.06 g, 7.64 mmol) and 1,1 equivalent of MOMCl (0.57 g, 7.08 mmol). The reaction mixture was heated to 40 oC for 2 hours. After completion of the reaction, the solvent was removed under reduced pressure, water was added and the product was extracted with ethyl acetate (3 × 30 mL).

The combined organic layer was washed with brine, dried over sodium sulfate, and evaporated under reduced pressure. The crude product was purified by column chromatography using hexanes/ethyl acetate (4:1) as eluent yielding the product as a buff powder, 0.61 g, 48%. 1H

NMR (300 MHz, CDCl3) δ 3.55 (s, 3H), 5.34 (s, 2H), 6.02 (s, 1H), 7.19 (d, J = 8.8 Hz, 1H),

7.77-7.85 (m, 2H).

148

Synthesis of 2-alkoxy-1-(methoxymethoxy)-4-nitrobenzene (3.15a-c)

To a solution of 3.14 (1.30-1.50 mmol) in dry acetonitrile (5 mL) was added 2 equivalents of potassium carbonate (2.60-3.00 mmol) and 3 equivalents of alkyl iodide (3.90-4.50 mmol). The mixture was heated in a sealed tube at 80 oC for 4-5 hours. After the reaction was complete, the solution was cooled and the solvent was removed under reduced pressure. Ice was added to precipitating the pure product, which was filtered as a white powder in 91-97% yield.

2-Methoxy-1-(methoxymethoxy)-4-nitrobenzene (3.15a) 0.29 g, yield 97%, starting from 0.28 g

1 3.14 (1.40 mmol). H NMR (300 MHz, CDCl3) δ 3.53 (s, 3H), 3.98 (s, 3H), 5.33 (s, 2H), 7.23 (d,

J = 8.9 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.88 (dd, J = 8.9, 2.6 Hz, 1H); 13C NMR (75 MHz,

CDCl3) δ 56.33, 56.62, 95.18, 106.91, 114.26, 117.49, 142.40, 149.42, 152.04.

2-Ethoxy-1-(methoxymethoxy)-4-nitrobenzene (3.15b) 0.28 g, yield 95%, starting from 0.26 g

1 3.14 (1.30 mmol). H NMR (300 MHz, CDCl3) δ 1.51 (t, J = 7.0 Hz, 3H), 3.53 (s, 3H), 4.19 (q, J

= 7.0 Hz, 2H), 5.32 (s, 2H), 7.22 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 2.6 Hz, 1H), 7.85 (dd, J = 9.0,

149

13 2.6 Hz, 1H); C NMR (75 MHz, CDCl3) δ 14.52, 56.57, 64.94, 95.21, 108.03, 114.81, 117.27,

142.43, 148.88, 152.29.

2-Isopropoxy-1-(methoxymethoxy)-4-nitrobenzene (3.15c) 0.33 g, yield 91%, starting from 0.30

1 g 3.14 (1.50 mmol). H NMR (300 MHz, CDCl3) δ 1.42 (d, J = 6.1 Hz, 6H), 3.52 (s, 3H), 4.64

(sep, J = 6.1 Hz, 1H), 5.30 (s, 2H), 7.21 (d, J = 9.0 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.85 (dd, J

13 = 9.0, 2.6 Hz, 1H); C NMR (75 MHz, CDCl3) δ 21.86, 56.52, 72.24, 95.17, 110.52, 115.34,

117.40, 142.45, 147.97, 153.25.

Synthesis of 2-alkoxy-4-nitrobenzene (3.16a-c)

To a cooled solution of 3.15a-c (1.1-1.31 mmol) in methanol/DCM (6:3) at 0 oC was added 4N

HCl (1 mL) and the mixture was stirred for 30 min. The mixture was then warmed to rt and allowed to stir for another 2 hours. After the reaction was complete, ethyl acetate (30 mL) was added and the product was extracted (2 × 30 mL) with ethyl acetate. The combined organic layer was washed with water and brine, dried over sodium sulfate, and evaporated under reduced

150 pressure. The crude product was purified by column chromatography affording the product in

82-89% yield.

2-Methoxy-4-nitrophenol (3.16a). Chromatography solvent: hexanes/DCM 1:1, yellow powder,

1 0.18 g, yield 82% starting from 0.28 g 3.15a (1.31 mmol); H NMR (300 MHz, CDCl3) δ 4.01 (s,

3H), 6.24 (s, 1H), 7.00 (d, J = 8.9 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.90 (dd, J = 8.9, 2.6 Hz,

13 1H); C NMR (75 MHz, CDCl3) δ 56.46, 106.34, 113.97, 118.62, 141.18, 146.12, 151.63.

2-Ethoxy-4-nitrophenol (3.16b). Chromatography solvent: hexanes/DCM 2:1 to 2:3, yellow

1 powder, 0.18 g, yield 89% starting from 0.25 g 3.15b (1.11 mmol); H NMR (400 MHz, CDCl3)

δ 1.53 (t, J = 7.0 Hz, 3H), 4.24 (q, J = 7.0 Hz, 2H), 6.29 (s, 1H), 7.00 (d, J = 8.9 Hz, 1H), 7.76

13 (d, J = 2.6 Hz, 1H), 7.89 (dd, J = 8.9, 2.6 Hz, 1H); C NMR (100 MHz, CDCl3) δ 14.57, 65.28,

106.99, 113.89, 118.45, 141.11, 145.39, 151.71.

2-Isopropoxy-4-nitrophenol (3.16c). Chromatography solvent: hexanes/DCM 2:1, yellow waxy

1 oil, 0.23 g, yield 90% starting from 0.31 g 3.15c (1.29 mmol); H NMR (300 MHz, CDCl3) δ

1.44 (t, J = 6.1 Hz, 6H), 4.73 (sep, J = 6.1 Hz, 1H), 6.33 (brs, 1H), 6.99 (d, J = 8.7 Hz, 1H), 7.76

13 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 8.7, 2.5 Hz, 1H); C NMR (75 MHz, CDCl3) δ 21.88, 72.61,

108.25, 114.01, 118.31, 141.07, 144.21, 152.44.

151

Synthesis of 1-((8-bromooctyl)oxy)-2-alkoxy-4-nitrobenzene (3.17a-c)

Compounds 3.17a-c were synthesized from 3.16a-c (0.92-1.11 mmol) following the general synthesis of compounds 3.9a-i.

1-((8-Bromooctyl)oxy)-2-methoxy-4-nitrobenzene (3.17a). Yellowish white powder, 0.33 g,

1 yield 91% starting from 0.17 g 3.16a (1.00 mmol); H NMR (300 MHz, CDCl3) δ 1.38-1.49 (m,

8H), 1.84-1.91 (m, 4H), 3.42 (t, J = 6.8 Hz, 2H), 3.95 (s, 3H), 4.11 (t, J = 6.7 Hz, 2H), 6.90 (d, J

= 9.1 Hz, 1H), 7.75 (d, J = 2.6 Hz, 1H), 7.90 (dd, J = 9.1, 2.5 Hz, 1H); 13C NMR (75 MHz,

CDCl3) δ 25.76, 28.03, 28.59, 28.82, 29.09, 32.71, 33.91, 56.31, 69.42, 106.69, 110.76, 117.76,

141.24, 149.08, 154.20.

1-((8-Bromooctyl)oxy)-2-ethoxy-4-nitrobenzene (3.17b). Yellowish white powder, 0.32 g, yield

1 92% starting from 0.17 g 3.16b (0.92 mmol); H NMR (300 MHz, CDCl3) δ 1.35-1.54 (m, 11H),

1.83-1.93 (m, 4H), 3.42 (t, J = 6.8 Hz, 2H), 4.10 (t, J = 6.7 Hz, 2H, overlapped), 4.16 (q, J = 7.0

Hz, 2H, overlapped), 6.90 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 2.6 Hz, 1H), 7.88 (dd, J = 8.9, 2.5 Hz,

152

13 1H); C NMR (75 MHz, CDCl3) δ 14.56, 25.77, 28.02, 28.62, 28.81, 29.08, 32.73, 33.90, 64.98,

69.42, 108.06, 111.08, 117.69, 141.23, 148.42, 154.53.

1-((8-Bromooctyl)oxy)-2-isopropoxy-4-nitrobenzene (3.17c). White powder, 0.36 g, yield 84%

1 starting from 0.22 g 3.16c (1.11 mmol); H NMR (300 MHz, CDCl3) δ 1.30-1.55 (m, 14H), 1.83-

1.92 (m, 4H), 3.42 (t, J = 6.8 Hz, 2H), 4.09 (t, J = 6.5 Hz, 2H), 4.58 (sep, J = 6.0 Hz, 1H), 6.90

(d, J = 8.9 Hz, 1H), 7.77 (d, J = 2.6 Hz, 1H), 7.89 (dd, J = 8.9, 2.5 Hz, 1H); 13C NMR (75 MHz,

CDCl3) δ 21.96, 25.79, 28.03, 28.65, 28.85, 29.07, 32.73, 33.89, 69.34, 72.61, 111.50, 111.58,

118.12, 141.19, 147.38, 155.81.

Synthesis of 1-(8(2-alkoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.18a-c)

Compounds 3.18a-c were synthesized from 3.17a-c (0.83-0.98 mmol) following the general synthesis of compounds 3.10a-m.

153

1-(8(2-Methoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.18a). Yellowish white powder, 0.23 g,

1 yield 74% starting from 0.32 g 3.17a (0.89 mmol); H NMR (300 MHz, CDCl3) δ 1.23-1.53 (m,

8H), 1.76-1.92 (m, 4H), 3.94 (s, 1H), 3.91-3.96 (m, 2H), 4.09 (t, J = 6.7 Hz, 2H), 6.87-6.91 (m,

2H), 7.05 (s, 1H), 7.46 (s, 1H), 7.74 (d, J = 2.6 Hz, 1H), 7.89 (dd, J = 8.9, 2.6 Hz, 1H); 13C NMR

(75 MHz, CDCl3) δ 25.67, 26.45, 28.81, 28.94, 29.10, 31.01, 46.97, 56.30, 69.37, 106.70,

110.77, 117.75, 118.72, 129.40, 137.05, 141.25, 149.06, 154.16

1-(8(2-Ethoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.18b). Yellowish white powder, 0.22 g,

1 yield 73% starting from 0.31 g 3.17b (0.83 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.53 (m,

11H), 1.74-1.93 (m, 4H), 3.94 (t, J = 7.1 Hz, 2H), 4.09 (t, J = 6.7 Hz, 2H), 4.16 (q, J = 7.0 Hz,

2H), 6.87-6.93 (m, 2H), 7.06 (s, 1H), 7.46 (s, 1H), 7.73 (d, J = 2.6 Hz, 1H), 7.88 (dd, J = 8.9, 2.6

13 Hz, 1H); C NMR (75 MHz, CDCl3) δ 14.55, 25.77, 26.46, 28.81, 28.97, 29.10, 31.02, 46.99,

64.97, 69.37, 108.06, 111.10, 117.68, 118.72, 129.40, 137.04, 141.25, 148.42, 154.50.

1-(8(2-Isopropoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.18c). Yellow oil, 0.33 g, yield 90%

1 starting from 0.38 g 3.17c (0.98 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.51 (m, 14H), 1.73-

1.89 (m, 4H), 3.93 (t, J = 7.1 Hz, 2H), 4.06 (t, J = 6.6 Hz, 2H), 4.56 (sep, J = 6.1 Hz, 1H), 6.87-

6.92 (m, 2H), 7.05 (s, 1H), 7.45 (s, 1H), 7.76 (d, J = 2.7 Hz, 1H), 7.87 (dd, J = 9.0, 2.7 Hz, 1H);

13 C NMR (75 MHz, CDCl3) δ 21.94, 25.79, 26.45, 28.83, 28.99, 29.08, 31.02, 46.98, 69.29,

72.60, 111.49, 115.59, 118.11, 118.73, 129.36, 137.03, 141.88, 147.35, 155.79.

154

Synthesis of 4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-alkoxyaniline (3.19a-c)

Compounds 3.19a-c were synthesized from 3.18a-c (0.49-0.86 mmol) following the general synthesis of compounds 3.11a-m.

4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-methoxyaniline (3.19a). Orange oil, 0.27 g, yield 99%

1 starting from 0.30 g 3.18a (0.86 mmol); H NMR (300 MHz, CDCl3) δ 1.21-1.50 (m, 8H), 1.70-

1.84 (m, 4H), 3.23 (brs, 2H), 3.81 (s, 3H), 3.89-3.95 (m, 4H), 6.21 (dd, J = 8.3, 2.6 Hz, 1H), 6.30

(d, J = 2.6 Hz, 1H), 6.72 (d, J = 8.3 Hz, 1H), 6.90 (t, J = 1.1 Hz, 1H), 7.05 (s, 1H), 7.46 (s, 1H).

4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-ethoxyaniline (3.19b). Brown oil, 0.16 g, yield 98%

1 starting from 0.18 g 3.18b (0.49 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.51 (m, 11H), 1.74-

1.85 (m, 4H), 2.18 (brs, 2H), 3.89-3.95 (m, 4H), 3.99-4.07 (m, 2H), 6.22 (dd, J = 8.4, 2.7 Hz,

1H), 6.31 ((d, J = 2.7 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.91 (s, 1H), 7.06 (s, 1H), 7.47 (s, 1H).

155

1-((8-(1H-imidazol-1-yl)octyl)oxy)-3-isopropoxyaniline (3.19c). Brown oil, 0.27 g, yield 98%

1 starting from 0.30 g 3.18c (0.79 mmol); H NMR (300 MHz, CDCl3) δ 1.23-1.49 (m, 14H), 1.68-

1.79 (m, 4H), 3.27 (brs, 2H), 3.86-3.95 (m, 4H), 4.43 (sep, J = 6.1 Hz, 1H), 6.23 (dd, J = 8.4, 2.7

Hz, 1H), 6.32 (d, J = 2.7 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.90 (s, 1H), 7.05 (s, 1H), 7.45 (s,

1H).

Synthesis of N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)3-alkoxyphenyl) picolinimidamide (3.20a-c)

Compounds 3.20a-c were synthesized from 3.19a-c (0.45-0.69 mmol) following the general synthesis of compounds 3.12a-n.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)3-methoxyphenyl) picolinimidamide (3.20a). Yellowish white powder, 130 mg, yield 45% starting from 220 mg 3.19a (0.69 mmol); mp 117-119 °C; 1H

NMR (400 MHz, CDCl3) δ 1.26-1.53 (m, 8H), 1.76-1.88 (m, 4H), 3.86 (s, 3H), 3.94 (t, J = 7.1

156

Hz, 2H), 4.01 (t, J = 6.9 Hz, 2H), 5.95 (brs, 2H), 6.57 (dd, J = 8.4, 2.2 Hz, 1H), 6.64 (d, J = 2.2

Hz, 1H), 6.89-6.92 (m, 2H), 7.07 (s, 1H), 7.41 (ddd, J = 7.4, 4.8, 1.1 Hz, 1H), 7.47 (s, 1H), 7.83

(td, J = 7.7, 1.6 Hz, 1H), 8.43 (d, J = 8.1 Hz, 1H), 8.59 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H); 13C NMR

(100 MHz, CDCl3) δ 25.89, 26.48, 28.98, 29.19, 29.27, 31.06, 47.02, 55.90, 69.47, 106.33,

112.67, 114.49, 118.74, 121.48, 125.13, 129.41, 136.79, 137.07, 143.35, 144.49, 147.88, 150.46,

+ 151.59, 153.09; HRMS (ESI) m/z (M +H) calcd for C24H32N5O2, 422.25505; found, 422.25616;

Anal. Calcd for C24H31N5O2: C, 68.38; H, 7.41; N, 16.61. Found: C, 68.18; H, 7.46; N, 16.38.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)3-ethoxyphenyl) picolinimidamide (3.20b). Yellowish white powder, 65 mg, yield 33% starting from 150 mg 3.19b (0.45 mmol); mp 100-102 °C; 1H

NMR (400 MHz, CDCl3) δ 1.26-1.53 (m, 11H), 1.76-1.87 (m, 4H), 3.94 (t, J = 7.1 Hz, 2H), 4.00

(t, J = 6.7 Hz, 2H), 4.09 (q, J = 7.0 Hz, 2H), 5.88 (brs, 2H), 6.56 (d, J = 8.5 Hz, 1H), 6.63 (s,

1H), 6.89-6.93 (m, 2H), 7.07 (s, 1H), 7.40 (ddd, J = 7.4, 4.8, 1.1 Hz, 1H), 7.47 (s, 1H), 7.82 (td, J

= 7.7, 1.7 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.59 (d, J = 4.8 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 14.91, 25.92, 26.49, 29.01, 29.19, 29.34, 31.07, 47.02, 64.38, 69.85, 107.90, 112.88,

115.55, 118.74, 121.46, 125.09, 129.41, 136.76, 137.07, 143.74, 144.83, 147.86, 149.99, 151.65,

+ 152.95; HRMS (ESI) m/z (M +H) calcd for C25H34N5O2, 436.27070; found, 436.27029; Anal.

Calcd for C25H33N5O2: C, 68.94; H, 7.64; N, 16.08. Found: C, 69.09; H, 7.54; N, 16.03.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)3-isopropoxyphenyl) picolinimidamide (3.20c). Yellowish white powder, 90 mg, yield 31% starting from 220 mg 3.19c (0.63 mmol); mp 88-91 °C; 1H

NMR (400 MHz, CDCl3) δ 1.30-1.42 (m, 12H), 1.44-1.54 (m, 2H), 1.77-1.84 (m, 4H), 3.94 (t, J

= 7.2 Hz, 2H), 3.99 (t, J = 6.5 Hz, 2H), 4.49 (sep, J = 6.0 Hz, 1H), 5.90 (brs, 2H), 6.59 (d, J = 8.2

157

Hz, 1H), 6.66 (s, 1H), 6.90-6.93 (m, 2H), 7.07 (s, 1H), 7.40 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 7.47

(s, 1H), 7.82 (td, J = 7.7, 1.7 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.59 (ddd, J = 4.9, 1.6, 0.9 Hz,

13 1H); C NMR (100 MHz, CDCl3) δ 22.28, 25.96, 26.50, 29.04, 29.19, 29.40, 31.08, 47.01,

69.83, 71.92, 111.50, 113.96, 116.16, 118.73, 121.46, 125.07, 129.42, 136.74, 137.07, 143.74,

+ 146.22, 147.85, 148.96, 151.69, 152.90; HRMS (ESI) m/z (M +H) calcd for C26H36N5O2,

450.28635; found, 450.28671; Anal. Calcd for C26H35N5O2: C, 69.46; H, 7.85; N, 15.58. Found:

C, 69.19; H, 7.77; N, 15.28.

Synthesis of 4-fluoro-2-alkoxy-1-nitrobenzene (3.22a-c)

To a solution of 3.21 (0.50-0.55 g, 3.18-3.50 mmol) in dry DMF (5 mL) was added 1.5 equivalent of potassium carbonate (4.77-5.25 mmol) and 3 equivalent of alkyl iodide (9.54-10.50 mmol) and the mixture was heated in a sealed tube at 80 oC for 4-5 hours. After reaction completion, the solution was cooled down and the solvent was removed under reduced pressure.

The crude product was purified by column chromatography to afford the product 3.20a-c in 64-

82% yield.

158

4-Fluoro-2-methoxy-1-nitrobenzene (3.22a) [186]. Chromatography solvent: hexanes/DCM 3:1 yellow powder, 0.43 g, yield 79% starting from 0.50 g 3.21 (3.18 mmol); 1H NMR (300 MHz,

CDCl3) δ 3.97 (s, 3H), 6.74 (ddd, J = 9.1, 7.2, 2.4 Hz, 1H), 6.80 (dd, J = 10.1, 2.4 Hz, 1H), 7.97

(dd, J = 9.1, 6.0 Hz, 1H).

4-Fluoro-2-ethoxy-1-nitrobenzene (3.22b) [187].Chromatography solvent: hexanes/DCM 4:1 yellow powder, 0.53 g, yield 82% starting from 0.55 g 3.21 (3.50 mmol); 1H NMR (300 MHz,

CDCl3) δ 1.51 (t, J = 6.9 Hz, 3H). 4.18 (q, J = 6.9 Hz, 2H), 6.72 (ddd, J = 9.1, 7.4, 2.5 Hz, 1H),

6.78 (dd, J = 10.3, 2.5 Hz, 1H), 7.96 (dd, J = 9.1, 6.0 Hz, 1H).

4-Fluoro-2-isopropoxy-1-nitrobenzene (3.22c) [187]. Chromatography solvent: hexanes/DCM

4:1 yellow oil, 0.42 g, yield 64% starting from 0.52 g 3.21 (3.30 mmol); 1H NMR (300 MHz,

CDCl3) δ 1.43 (d, J = 6.0 Hz, 6H). 4.64 (sep, J = 6.0 Hz, 1H), 6.70 (ddd, J = 9.1, 7.4, 2.6 Hz,

1H), 6.78 (dd, J = 10.5, 2.6 Hz, 1H), 7.88 (dd, J = 9.1, 6.1 Hz, 1H).

159

Synthesis of 3-alkoxy-4-nitrophenol (3.23a-c)

3.23a-c were prepared according to a previously published procedure [168]. To a solution of

3.22a-c (1.30-1.80 mmol) in DMSO (5 mL) was added NaOH (0.5 g) dissolved in distilled water

(5 mL) and the mixture was stirred vigorously at 80 oC for 20 hours. After completion of the reaction, the solution was cooled and the pH was rendered acidic with 6N HCl. The product was extracted with ethyl acetate (3 × 30 mL), washed with water and brine and dried over sodium sulfate. The combined organic layer was evaporated under reduced pressure then purified by column chromatography using hexanes/ethyl acetate 2:1 as eluent to afford the pure product in

74-85% yield.

3-Methooxy-4-nitrophenol (3.23a) [188]. Yellow powder, 0.32 g, yield 83%, starting from 0.39

1 g 3.22a (2.27 mmol). H NMR (300 MHz, DMSO-d6) δ 3.86 (s, 3H), 6.46 (dd, J = 9.1, 2.4 Hz,

1H), 6.59 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 9.1 Hz, 1H), 10.87 (brs, 1H).

160

2-Ethoxy-1-(methoxymethoxy)-4-nitrobenzene (3.23b). Yellow powder, 0.36 g, yield 74%,

1 starting from 0.49 g 3.22b (2.64 mmol). H NMR (300 MHz, DMSO-d6) δ 1.34 (t, J = 6.9 Hz,

3H ), 4.12 (q, J = 6.9 Hz, 2H), 6.45 (dd, J = 8.9, 2.3 Hz, 1H), 6.58 (d, J = 2.3 Hz, 1H), 7.86 (d, J

13 = 8.9 Hz, 1H), 10.83 (brs, 1H); C NMR (75 MHz, DMSO-d6) δ 14.78, 65.23, 101.47, 107.91,

128.58, 131.56, 155.29, 164.21.

2-Isopropoxy-1-(methoxymethoxy)-4-nitrobenzene (3.23c). Yellow powder, 0.30 g, yield 85%,

1 starting from 0.36 g 3.22c (1.80 mmol). H NMR (300 MHz, DMSO-d6) δ 1.29 (d, J = 6.0 Hz,

6H ), 4.69 (sep, J = 6.0 Hz, 1H), 6.44 (dd, J = 9.0, 2.3 Hz, 1H), 6.60 (d, J = 2.3 Hz, 1H), 7.82 (d,

13 J = 9.0 Hz, 1H), 10.81 (brs, 1H); C NMR (75 MHz, DMSO-d6) δ 20.72, 72.11, 102.05, 107.00,

127.72, 132.87, 154.10, 163.41.

Synthesis of 4-((8-bromooctyl)oxy)-2-alkoxy-1-nitrobenzene (3.24a-c)

Compounds 3.24a-c were synthesized from 3.23a-c (1.32-2.06 mmol) following the general synthesis of compounds 3.9a-i.

161

4-((8-Bromooctyl)oxy)-2-methoxy-1-nitrobenzene (3.24a). Yellow powder, 0.67 g, yield 90%

1 starting from 0.35 g 3.23a (2.06 mmol); H NMR (300 MHz, CDCl3) δ 1.32-1.60 (m, 8H), 1.78-

1.90 (m, 4H), 3.42 (t, J = 6.8 Hz, 2H), 3.96 (s, 3H), 4.04 (t, J = 6.5 Hz, 2H), 6.48 (dd, J = 9.1, 2.5

13 Hz, 1H), 6.52 (d, J = 2.5 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H); C NMR (75 MHz, CDCl3) δ 25.81,

28.02, 28.61, 28.95, 29.08, 32.71, 33.90, 56.45, 68.73, 100.05, 105.14, 128.48, 132.78, 155.72,

164.40.

1-((8-Bromooctyl)oxy)-2-ethoxy-4-nitrobenzene (3.24b). Yellow powder, 0.51 g, yield 76%

1 starting from 0.33 g 3.23b (1.80 mmol); H NMR (300 MHz, CDCl3) δ 1.3-1.53 (m, 8H), 1.50 (t,

J = 7.0 Hz, 3H, overlapped) 1.77-1.93 (m, 4H), 3.43 (t, J = 6.8 Hz, 2H), 4.02 (t, J = 6.5 Hz, 2H),

4.16 (q, J = 7.0 Hz, 2H), 6.48 (dd, J = 9.0, 2.5 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 7.96 (d, J = 9.0

13 Hz, 1H); C NMR (75 MHz, CDCl3) δ 14.52, 25.82, 28.03, 28.62, 28.96, 29.10, 32.72, 33.94,

65.35, 68.67, 100.89, 105.12, 128.26, 133.10, 155.02, 164.17.

1-((8-Bromooctyl)oxy)-2-isopropoxy-4-nitrobenzene (3.24c). White powder, 0.48 g, yield 94%

1 starting from 0.26 g 3.23c (1.32 mmol); H NMR (400 MHz, CDCl3) δ 1.32-1.52 (m, 14H), 1.77-

1.91 (m, 4H), 3.42 (t, J = 6.8 Hz, 2H), 4.01 (t, J = 6.5 Hz, 2H), 4.64 (sep, J = 6.0 Hz, 1H), 6.47

(dd, J = 9.0, 2.5 Hz, 1H), 6.52 (d, J = 2.5 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 21.96, 25.79, 28.03, 28.65, 28.85, 29.07, 32.73, 33.89, 69.34, 72.61, 111.50, 111.58,

118.12, 141.19, 147.38, 155.81.

162

Synthesis of 1-(8(3-alkoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.25a-c)

Compounds 3.25a-c were synthesized from 3.24a-c (0.85-1.38 mmol) following the general synthesis of compounds 3.10a-m.

1-(8(3-Methoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.25a). Yellow powder, 0.28 g, yield 81%

1 starting from 0.36 g 3.24a (1.00 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.52 (m, 8H), 1.76-

1.86 (m, 4H), 3.95 (s, 3H), 3.91-3.97 (m, 2H, overlapped), 4.02 (t, J = 6.5 Hz, 2H), 6.49 (dd, J =

9.1, 2.4 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 6.91 (s, 1H), 7.06 (s, 1H), 7.46 (s, 1H), 8.00 (d, J = 9.1

13 Hz, 1H); C NMR (75 MHz, CDCl3) δ 25.81, 26.45, 28.95, 28.97, 29.12, 31.02, 46.97, 56.46,

68.67, 100.02, 105.13, 118.72, 128.48, 129.44, 132.77, 137.07, 155.71, 164.37.

1-(8(3-Ethoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.25b). Yellow powder, 0.25 g, yield 81%

1 starting from 0.32 g 3.24b (0.85 mmol); H NMR (300 MHz, CDCl3) δ 1.28-1.53 (m, 11H), 1.73-

1.93 (m, 4H), 3.94 (t, J = 7.1 Hz, 2H), 4.09 (t, J = 6.7 Hz, 2H), 4.16 (q, J = 7.0 Hz, 2H), 6.49 (dd,

163

J = 9.1, 2.4 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 6.91 (s, 1H), 7.06 (s, 1H), 7.46 (s, 1H), 8.00 (d, J =

13 9.1 Hz, 1H); C NMR (75 MHz, CDCl3) δ 14.55, 25.77, 26.46, 28.81, 28.97, 29.10, 31.02,

46.99, 64.97, 69.37, 108.06, 111.10, 117.68, 118.72, 129.40, 137.04, 141.25, 148.42, 154.50

1-(8(2-Isopropoxy-4-nitrophenoxy)octyl)-1H-imidazole (3.25c). Yellow oil, 0.35 g, yield 79%

1 starting from 0.46 g 3.24c (1.18 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.51 (m, 14H), 1.73-

1.89 (m, 4H), 3.93 (t, J = 7.1 Hz, 2H), 4.06 (t, J = 6.6 Hz, 2H), 4.56 (sep, J = 6.1 Hz, 1H), 6.49

(dd, J = 9.1, 2.4 Hz, 1H), 7.52 (d, J = 2.4 Hz, 1H), 6.91 (s, 1H), 7.06 (s, 1H), 7.46 (s, 1H), 8.00

13 (d, J = 9.1 Hz, 1H); C NMR (75 MHz, CDCl3) δ 21.94, 25.79, 26.45, 28.83, 28.99, 29.08,

31.02, 46.98, 69.29, 72.60, 111.49, 115.59, 118.11, 118.73, 129.36, 137.03, 141.88, 147.35,

155.79.

164

Synthesis of 4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-alkoxyaniline (3.26a-c)

Compounds 3.26a-c were synthesized from 3.25a-c (0.49-0.86 mmol) following the general synthesis of compounds 3.11a-m.

4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-methoxyaniline (3.26a). yellow oil, 0.26 g, yield 95%

1 starting from 0.30 g 3.25a (0.86 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.50 (m, 8H), 1.70-

1.84 (m, 4H), 3.12 (brs, 2H), 3.83 (s, 3H), 3.86-3.95 (m, 4H), 6.34 (dd, J = 8.4, 2.4 Hz, 1H), 6.45

(d, J = 2.4 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 6.91 (t, J = 1.1 Hz, 1H), 7.06 (s, 1H), 7.47 (s, 1H).

4-((8-(1H-imidazol-1-yl)octyl)oxy)-3-ethoxyaniline (3.26b). Brown oil, 0.21 g, yield 95%

1 starting from 0.24 g 3.25b (0.66 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.50 (m, 11H), 1.69-

1.84 (m, 4H), 3.06 (brs, 2H), 3.88 (t, J = 6.6 Hz, 2H), 3.93 (t, J = 7.1 Hz, 2H), 4.04 (q, J = 6.9

Hz, 2H), 6.34 (dd, J = 8.3, 2.5 Hz, 1H), 6.46 (d, J = 2.5 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.91

(s, 1H), 7.06 (s, 1H), 7.47 (s, 1H).

165

1-((8-(1H-imidazol-1-yl)octyl)oxy)-3-isopropoxyaniline (3.26c). Brown oil, 0.24 g, yield 100%

1 starting from 0.26 g 3.25c (0.69 mmol); H NMR (300 MHz, CDCl3) δ 1.25-1.50 (m, 14H), 1.69-

1.83 (m, 4H), 3.21 (brs, 2H), 3.87 (t, J = 6.7 Hz, 2H), 3.92 (t, J = 7.1 Hz, 2H), 4.50 (sep, J = 6.1

Hz, 1H), 6.34 (dd, J = 8.5, 2.6 Hz, 1H), 6.46 (d, J = 2.6 Hz, 1H), 6.64 (d, J = 8.5 Hz, 1H), 6.90

(s, 1H), 7.06 (s, 1H), 7.47 (s, 1H).

Synthesis of N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)2-alkoxyphenyl) picolinimidamide (3.27a-c)

Compounds 3.27a-c were synthesized from 3.26a-c (0.54-0.63 mmol) following the general synthesis of compounds 3.12a-n.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)3-methoxyphenyl) picolinimidamide (3.27a). Yellowish white powder, 120 mg, yield 45% starting from 200 mg 3.26a (0.63 mmol); mp 109-112°C; 1H

NMR (300 MHz, CDCl3) δ 1.28-1.53 (m, 8H), 1.73-1.85 (m, 4H), 3.82 (s, 3H), 3.91-4.00 (m,

166

4H), 5.88 (brs, 2H), 6.52 (dd, J = 8.4, 2.4 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 6.92 (s, 1H), 6.90-

7.01 (brs, 1H, overlapped), 7.07 (s, 1H), 7.36-7.43 (m, 1H), 7.47 (m, 1H),7.78-7.86 (m, 1H), 8.48

+ (d, J = 7.8 Hz, 1H). 8.58 (d, J = 4.5 Hz, 1H); HRMS (ESI) m/z (M +H) calcd for C24H32N5O2,

422.25505; found, 422.25452; Anal. Calcd for C24H31N5O2: C, 68.38; H, 7.41; N, 16.61. Found:

C, 68.47; H, 7.54; N, 16.61.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)3-ethoxyphenyl) picolinimidamide (3.27b). Buff powder,

100 mg, yield 42% starting from 180 mg 3.26b (0.54 mmol); mp 86-89°C; 1H NMR (700 MHz,

CDCl3) δ 1.31-1.39 (m, 9H), 1.45-1.50 (m, 2H), 1.76-1.83 (m, 4H), 3.93-3.97 (m, 4H), 4.05 (q, J

= 6.9 Hz, 2H), 6.04 (brs, 2H), 6.53 (dd, , J = 8.5, 2.5 Hz, 1H), 6.58 (d, , J = 2.5 Hz, 1H), 6.92 (s,

1H), 7.00 (brs, 1H), 7.07 (s, 1H), 7.39-7.42 (m, 1H), 7.47 (s, 1H), 7.83 (td, J = 7.8, 1.6 Hz, 1H),

13 8.49 (d, J = 7.7 Hz, 1H), 8.59 (d, , J = 4.6 Hz, 1H); C NMR (175 MHz, CDCl3) δ 14.89, 25.98,

26.49, 29.01, 29.20, 29.32, 31.07, 47.02, 64.42, 68.16, 102.04, 105.95, 118.76, 121.89, 123.36,

125.09, 129.44, 136.83, 137.09, 147.85, 151.03, 151.38, 153.42, 156.38; HRMS (ESI) m/z (M

+ +H) calcd for C25H34N5O2, 436.27070; found, 436.27139; Anal. Calcd for C25H33N5O2: C,

68.94; H, 7.64; N, 16.08. Found: C, 68.66; H, 7.65; N, 15.86.

N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)3-isopropoxyphenyl) picolinimidamide (3.27c). Yellow powder, 92 mg, yield 34% starting from 210 mg 3.26c (0.60 mmol); mp 82-85 °C; 1H NMR

(700 MHz, CDCl3) δ 1.26 (d, J = 3.2 Hz, 6H), 1.30-1.39 (m, 6H), 1.44-1.49 (m, 2H), 1.75-1.82

(m, 4H), 3.94 (t, J = 6.8 Hz, 4H), 4.45 (sep, J = 5.9 Hz, 1H), 5.80 (brs, 2H), 6.56 (dd, , J = 8.5,

2.6 Hz, 1H), 6.57 (d, , J = 2.6 Hz, 1H), 6.92 (s, 1H), 6.88-6.94 (brs, 1H, overlapped), 7.07 (s,

1H), 7.38-7.40 (m, 1H), 7.47 (s, 1H), 7.80-7.83 (m, 1H), 8.45 (brs, 1H), 8.508(d, , J = 3.8 Hz,

167

13 1H) C NMR (175 MHz, CDCl3) δ 22.29, 25.99, 26.51, 29.03, 29.22, 29.33, 31.08, 47.02,

68.18, 71.99, 105.75, 107.60, 118.76, 121.68, 123.60, 124.89, 129.44, 133.67, 136.71, 137.09,

+ 147.82, 149.68, 151.90, 152.47, 155.97; HRMS (ESI) m/z (M +H) calcd for C26H36N5O2,

450.28635; found, 450.28778; Anal. Calcd for C26H35N5O2: C, 69.46; H, 7.85; N, 15.58. Found:

C, 69.40; H, 7.90; N, 15.37.

Synthesis of biphenoxyalkyl AIA azole intermediates. (3.29a,b)

Compounds 3.29a,b were synthesized from 3.28 (9.08-11.36 mmol) following the general synthesis of compounds 3.6a-i. except that gentle heating overnight was carried out at 50 oC instead of 80 oC and further crystallization of the product from methanol was performed after column chromatography.

2-(4-(4-Bromobutoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.29a) [189].

Chromatography solvent: hexanes/ethyl acetate 10:1, white crystalline solid, 2.42 g, yield 75%

168

1 starting from 2.00 g 3.28 (9.08 mmol); H NMR (400 MHz, CDCl3) δ 1.35 (s, 12H), 1.93-2.01

(m, 2H), 2.05-2.14 (m, 2H), 3.51 (t, J = 6.4 Hz, 2H), 4.04 (t, J = 6.4 Hz, 2H), 6.90 (d, J = 8.6

Hz, 2H), 7.76 (d, J = 8.6 Hz, 2H).

2-(4-((6-Bromohexyl)oxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.29b) [190].

Chromatography solvent: hexanes/DCM 2:1 to 1:3, white crystalline solid, 3.40 g, yield 78%

1 starting from 2.50 g 3.28 (11.36 mmol); H NMR (400 MHz, CDCl3) δ 1.35 (s, 12H), 1.49-1.57

(m, 4H), 1.78-1.86 (m, 2H), 1.88-1.96 (m, 2H), 3.44 (t, J = 6.8 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H),

6.90 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 8.6 Hz, 2H).

Synthesis of biphenoxyalkyl AIA azole intermediates. (3.30a,b)

Compounds 3.30a,b were synthesized from 3.29a,b (3.88-8.35 mmol) following the general synthesis of compounds 3.10a-m except that gentle heating overnight at 50 oC was carried out

169 instead of 80 oC and further crystallization of the product from hexanes/ethyl acetate was performed after column chromatography.

1-(4-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyl)-1H-imidazole (3.30a).

Chromatography solvent: DCM/MeOH 10:0.4, white crystalline solid, 0.75 g, yield 56% starting

1 from 1.38 g 3.29a (3.88 mmol); H NMR (400 MHz, CDCl3) δ 1.35 (s, 12H), 1.76-1.85 (m, 2H),

1.96-2.05 (m, 2H), 3.99-4.07 (m, 4H), 6.88 (d, J = 8.4 Hz, 2H), 6.95 (s, 1H), 7.09 (s, 1H), 7.51

13 (s, 1H), 7.76 (d, J = 8.4 Hz, 2H); C NMR (100 MHz, CDCl3) δ 24.86, 26.24, 28.07, 46.73,

66.86, 83.58, 113.76, 118.74, 129.60, 136.56, 137.12, 161.29.

1-(6-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)hexyl)-1H-imidazole (3.30b).

Chromatography solvent: hexanes/DCM 2:1 to 1:3, white crystalline solid, 1.9 g, yield 61%

1 starting from 3.20 g 3.29b (8.35 mmol); H NMR (300 MHz, CDCl3) δ 1.30-45 (m, 14H), 1.46-

1.57 (m, 2H), 1.72-1.88 (m, 4H), 3.91-4.01 (m, 4H), 6.85-6.90 (m, 2H), 6.91 (t, J = 1.2 Hz, 1H),

13 7.07 (t, J = 0.9 Hz, 1H), 7.47 (s, 1H), 7.72-7.79 (m, 2H); C NMR (100 MHz, CDCl3) δ 24.84,

25.59, 26.31, 28.99, 31.01, 46.89, 67.36, 83.52, 113.82, 118.71, 129.49, 136.50, 137.05, 161.57.

170

Synthesis of biphenoxyalkyl AIA azole intermediates. (3.31a-f)

To a degassed solution of 3.31a,b (0.86-1.02 mmol) in DMF (8 mL) in a three necked flask was added 1.2 equivalents of halobenzene (1.03-1.22 mmol) and 2 equivalents of potassium carbonate as a 2M solution (1.72-2.04 mmol) and 6 mol% of Pd(PPh3)4 catalyst (60-71 mg) and the mixture was heated at 100°C under nitrogen atmosphere overnight. After the reaction was complete, the solvent was removed under reduced pressure, water was added (30 mL) and the product was extracted with DCM (3 × 30 mL). The combined organic layer was dried over sodium sulfate, and filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography affording the product in 67-89% yield.

1-(4-((4ʹ-Nitro-[1,1ʹ-biphenyl]-4-yl)oxy)butyl)-1H-imidazole (3.31a). Chromatography solvent:

DCM/ethyl acetate 5:1, yellow powder, 0.25 g, yield 73% starting from 0.35 g 3.30a (1.02

1 mmol); H NMR (400 MHz, CDCl3) δ 1.80-1.90 (m, 2H), 2.00-2.09 (m, 2H), 4.01-4.10 (m, 4H),

171

6.97 (s, 1H), 7.00 (d, J = 8.8 Hz, 2H), 7.10 (s, 1H), 7.53 (s, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.70

13 (d, J = 8.8 Hz, 2H), 8.30 (d, J = 8.8 Hz, 2H); C NMR (100 MHz, CDCl3) δ 26.30, 28.04, 46.73,

67.29, 115.07, 118.73, 124.15, 127.08, 128.62, 129.65, 131.28, 137.11, 146.60, 147.11, 159.60.

1-(6-((4ʹ-Nitro-[1,1ʹ-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole (3.31b). Chromatography solvent: hexanes/ethanol 5:1 on trimethylamine neutralized silica, yellow powder, 0.23 g, yield 73%

1 starting from 0.32 g 3.30b (0.86 mmol); H NMR (400 MHz, CDCl3) δ 1.36-1.45 (m, 2H), 1.50-

1.61 (m, 2H), 1.76-1.90 (m, 4H), 3.95-4.07 (m, 4H), 6.94 (s, 1H), 6.99-7.03 (m, 2H), 7.09 (s,

1H), 7.54 (s, 1H), 7.56-7.62 (m, 2H), 7.68-7.72 (m, 2H), 8.8.25-8.30 (m, 2H); 13C NMR (100

MHz, CDCl3) δ 25.62, 26.31, 29.01, 30.99, 46.99, 67.79, 115.10, 118.80, 124.14, 127.04,

128.56, 129.24, 130.98, 137.02, 146.54, 147.20, 159.88.

1-(4-((3ʹ-Methoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-yl)oxy)butyl)-1H-imidazole (3.31c).

Chromatography solvent: Hexanes/ethanol 5:1 on trimethylamine neutralized silica, yellow oil,

1 0.29 g, yield 77% starting from 0.35 g 3.30a (1.02 mmol); H NMR (300 MHz, CDCl3) δ 1.78-

1.87(m, 2H), 1.98-2.08 (m, 2H), 4.03 (s, 3H), 4.00-4.09 (m, 4H, overlapped), 6.94-7.02 (m, 3H),

7.08 (s, 1H), 7.16-7.24 (m, 2H), 7.49-7.59 (m, 3H), 7.94 (d, J = 8.3 Hz, 1H); 13C NMR (75

MHz, CDCl3) δ 26.28, 28.01, 46.72, 56.53, 67.30, 111.51, 114.99, 118.48, 118.73, 126.51,

128.54, 129.63, 131.64, 137.10, 137.88, 147.38, 153.61, 159.55.

1-(6-((3ʹ-Methoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole (3.31d).

Chromatography solvent: DCM/MeOH 10:0.2, yellow oil, 0.31 g, yield 83% starting from 0.35 g

1 3.30b (0.94 mmol); H NMR (300 MHz, CDCl3) δ 1.33-1.46 (m, 2H), 1.47-1.59 (m, 2H), 1.77-

1.88 (m, 4H), 4.02 (s, 3H), 3.92-4.04 (m, 4H, overlapped), 6.92 (s, 1H), 6.96-7.01 (m, 2H), 7.06

172

(s, 1H), 7.15-7.21 (m, 2H), 7.47 (s, 1H), 7.50-7.56 (m, 2H), 7.95 (d, J = 8.4 Hz, 1H); 13C NMR

(100 MHz, CDCl3) δ 25.60, 26.30, 28.99, 30.99, 46.89, 56.53, 67.81, 111.47, 115.03, 118.45,

118.75, 126.50, 128.47, 129.47, 131.34, 137.07, 137.81, 147.48, 153.62, 159.83.

1-(4-((3ʹ-Isopropoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-yl)oxy)butyl)-1H-imidazole (3.31e).

Chromatography solvent: Hexanes/ethanol 5:1 on trimethylamine neutralized silica, yellow oil,

1 0.27 g, yield 67% starting from 0.35 g 3.30a (1.02 mmol); H NMR (300 MHz, CDCl3) δ 1.44 (d,

J = 6.1 Hz, 6H), 1.78-1.87(m, 2H), 1.98-2.08 (m, 2H), 4.01-4.09 (m, 4H), 4.76 (sep, J = 6.1 Hz,

1H), 6.95-7.00 (m, 3H), 7.09 (s, 1H), 7.14 (dd, J = 8.4, 1.7 Hz, 1H), 7.19 (d, J = 1.7 Hz, 1H),

13 7.48-7.55 (m, 3H), 7.87 (d, J = 8.4 Hz, 1H); C NMR (75 MHz, CDCl3) δ 21.96, 26.28, 28.02,

46.73, 67.29, 72.83, 114.40, 114.96, 118.46, 118.74, 126.22, 128.49, 129.62, 131.80, 137.10,

139.37, 146.76, 151.90, 159.43.

1-(6-((3ʹ-Isopropoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole (3.31f).

Chromatography solvent: DCM/MeOH 10:0.2, yellow oil, 0.36 g, yield 89% starting from 0.35 g

1 3.30b (0.94 mmol); H NMR (400 MHz, CDCl3) δ 1.35-1.48 (m, 8H), 1.50-1.58 (m, 2H), 1.78-

1.89 (m, 4H), 3.94-4.04 (m, 4H), 4.77 (sep, J = 6.1 Hz, 1H), 6.93 (s, 1H), 6.99 (d, J = 8.6 Hz,

2H), 7.08 (s, 1H), 7.15 (dd, J = 8.5, 1.6 Hz, 1H), 7.21 (d, J = 1.6 Hz, 1H), 7.49 (s, 1H), 7.52 (d,

13 J = 8.6 Hz, 2H), 7.88 (d, J = 8.5 Hz, 1H); C NMR (100 MHz, CDCl3) δ 21.96, 25.62, 26.31,

29.01, 31.01, 46.91, 67.80, 72.81, 114.36, 115.00, 118.44, 118.76, 126.24, 128.44, 129.47,

131.52, 137.07, 139.31, 146.87, 151.92, 159.70.

173

Synthesis of biphenoxyalkyl AIA azole intermediates. (3.31g-j)

A three necked flask was charged with 1-bromo-2-methoxy-4-nitrobenzene (1.03-1.29 mmol) or

1-bromo-2-isopropoxy-4-nitrobenzene (1.03 mmol), 1.2 equivalent of 3.30a,b, 2 equivalent of potassium carbonate (2.06-2.58 mmol), and 6 mol% of Pd(dppf)Cl2 catalyst (54-57 mg).

Degassed DMSO (10 mL) and degassed distilled water (1 mL) were added and the mixture was heated at 100 °C under nitrogen atmosphere overnight. After completion of the reaction, the solvent was removed under reduced pressure, water was added (30 mL) and the product was extracted with DCM (3 × 30 mL). The combined organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified by column chromatography affording the product in 79-86% yield.

1-(4-((2ʹ-Methoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-yl)oxy)butyl)-1H-imidazole (3.31g).

Chromatography solvent: Hexanes/ethanol 100:8 on trimethylamine neutralized silica, yellow

174 powder, 0.30 g, yield 79% starting from 0.24 g 1-bromo-2-methoxy-4-nitrobenzene (1.03 mmol)

1 and 0.42 g 3.30a (1.24 mmol); H NMR (400 MHz, CDCl3) δ 1.80-1.86 (m, 2H), 2.01-2.08 (m,

2H), 3.93 (s, 3H), 4.02-4.10 (m, 4H), 6.95-7.00 (m, 3H), 7.10 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H),

7.48-7.52 (m, 2H), 7.55 (s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.91 (dd, J = 8.5, 2.1 Hz, 1H); 13C

NMR (100 MHz, CDCl3) δ 26.28, 28.08, 46.80, 56.09, 67.17, 106.19, 114.23, 116.16, 118.80,

128.84, 129.44, 130.62, 130.73, 137.00, 137.08, 147.62, 156.70, 158.85.

1-(6-((2ʹ-Methoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole (3.31h).

Chromatography solvent: Hexanes/ethanol 100:8 on triethylamine neutralized silica, yellow powder, 0.40 g, yield 78% starting from 0.30 g of 1-bromo-2-methoxy-4-nitrobenzene (1.29

1 mmol) and 0.57 g of 3.30b (1.55 mmol); H NMR (300 MHz, CDCl3) δ 1.34-1.46 (m, 2H), 1.48-

1.59 (m, 2H), 1.77-1.90 (m, 4H), 3.93 (s, 3H), 3.95-4.04 (m, 4H), 6.93 (s, 1H), 6.95-6.99 (m,

2H), 7.08 (s, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.47-7.52 (m, 2H), 7.53 (s, 1H), 7.82 (d, J = 2.1 Hz,

13 1H), 7.90 (dd, J = 8.3, 2.1 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.62, 26.92, 29.02, 30.99,

46.99, 56.07, 67.65, 106.20, 114.27, 116.15, 118.79, 128.55, 129.19, 130.60, 130.66, 136.99,

137.11, 147.57, 156.70, 159.14.

1-(4-((2ʹ-Isopropoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-yl)oxy)butyl)-1H-imidazole (3.31i).

Chromatography solvent: Hexanes/ethanol 10:0.6 to 10:0.9 on trimethylamine neutralized silica, waxy brown oil, 0.27 g, yield 67% starting from 0.27 g of 1-bromo-2-isopropoxy-4-nitrobenzene

1 (1.03 mmol) and 0.42 g 3.30a (1.24 mmol); H NMR (400 MHz, CDCl3) δ 1.36 (d, J = 6.1 Hz,

6H), 1.79-1.87 (m, 2H), 2.00-2.09 (m, 2H), 4.02-4.10 (m, 4H), 4.65 (sep, J = 6.1 Hz, 1H), 6.93-

6.99 (m, 3H), 7.11 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.50-7.54 (m, 2H), 7.56 (s, 1H), 7.81 (d, J

175

13 = 1.9 Hz, 1H), 7.87 (dd, J = 8.4, 1.7 Hz, 1H); C NMR (100 MHz, CDCl3) δ 21.78, 26.31,

28.10, 46.79, 67.13, 71.48, 108.87, 114.02, 115.88, 118.78, 129.26, 129.50, 130.76, 130.82,

137.11, 137.90, 147.46, 154.98, 158.70.

1-(6-((2ʹ-Isopropoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole (3.31j).

Chromatography solvent: Hexanes/ethanol 10:0.6 to 10:0.9 on trimethylamine neutralized silica, brown oil, 0.38 g, yield 87% starting from 0.27 g of 1-bromo-2-isopropoxy-4-nitrobenzene (1.03

1 mmol) and 0.46 g 3.30b (1.24 mmol); H NMR (300 MHz, CDCl3) δ 1.35-1.47 (m, 8H), 1.49-

1.59 (m, 2H), 1.77-1.89 (m, 4H), 3.94-4.04 (m, 4H), 4.63 (sep, J = 6.1 Hz, 1H), 6.92-6.97 (m,

3H), 7.07 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.48-7.53 (m, 3H), 7.80 (d, J = 2.1 Hz, 1H), 7.86

13 (dd, J = 8.4, 2.1 Hz, 1H); C NMR (75 MHz, CDCl3) δ 21.78, 25.64, 26.32, 29.07, 31.01, 46.93,

67.62, 71.48, 108.92, 114.05, 115.88, 118.77, 128.97, 129.40, 130.69, 130.79, 137.07, 138.04,

147.40, 154.97, 158.97.

176

Synthesis of biphenoxyalkyl AIA azole intermediates. (3.32a-j)

Compounds 3.32a-j were synthesized from 3.31a-j (0.54-0.82 mmol) following the general synthesis of compounds 3.8a-m.

4'-(4-(1H-imidazol-1-yl)butoxy)-[1,1'-biphenyl]-4-amine (3.32a). Yellow oil, 0.19 g, yield 91%

1 starting from 0.23 g 3.31a (0.68 mmol); H NMR (300 MHz, CDCl3) δ 1.75-1.86 (m, 2H), 1.97-

2.07 (m, 2H), 3.68 (brs, 2H), 3.98-4.08 (m, 4H), 6.72-6.78 (m, 2H), 6.89-6.97 (m, 3H), 7.09 (s,

13 1H), 7.34-7.38 (m, 2H), 7.43-7.49 (m, 2H), 7.52 (s, 1H); C NMR (300 MHz, CDCl3) δ 26.34,

28.11, 46.75, 67.18, 114.65, 115.41, 118.74, 127.43, 127.58, 129.58, 131.23, 134.12, 137.11,

145.38, 157.55.

4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-[1,1'-biphenyl]-4-amine (3.32b). Yellow oil, 0.16 g, yield

1 87% starting from 0.20 g 3.31b (0.54 mmol); H NMR (400 MHz, CDCl3) δ 1.35-1.44 (m, 2H),

177

1.49-1.58 (m, 2H), 1.76-1.88 (m, 4H), 3.51 (brs, 2H), 3.94-4.01 (m, 4H), 6.74-6.77 (m, 2H),

6.90-6.96 (m, 3H), 7.08 (s, 1H), 7.36-7.40 (m, 2H), 7.43-7.48 (m, 2H), 7.50 (s, 1H); 13C NMR

(100 MHz, CDCl3) δ 25.65, 26.33, 29.11, 31.03, 46.95, 67.67, 114.68, 115.42, 118.79, 127.38,

127.57, 129.39, 131.33, 133.84, 137.06, 145.33, 157.82.

4'-(4-(1H-imidazol-1-yl)butoxy)-3-methoxy-[1,1'-biphenyl]-4-amine (3.32c). Buff powder, 0.17

1 g, yield 77% starting from 0.24 g 3.31c (0.65 mmol); H NMR (300 MHz, CDCl3) δ 1.76-1.87

(m, 2H), 1.98-2.08 (m, 2H), 3.86 (brs, 2H), 3.93 (s, 3H), 4.00-4.09 (m, 4H), 6.75-6.80 (m, 1H),

6.88-6.95 (m, 2H), 6.96 (s, 1H), 6.98-7.03 (m, 2H), 7.09 (s, 1H), 7.44-7.50 (m, 2H), 7.52 (s,

13 1H); C NMR (75 MHz, CDCl3) δ 26.33, 28.12, 46.78, 55.54, 67.17, 109.14, 114.62, 115.14,

118.77, 119.37, 127.61, 129.54, 131.54, 134.49, 135.18, 137.11, 147.52, 157.59.

4'-(6-((1H-imidazol-1-yl)hexyl)oxy)-3-methoxy-[1,1'-biphenyl]-4-amine (3.32d). White powder,

1 0.19 g, yield 76% starting from 0.27 g 3.31d (0.68 mmol); H NMR (300 MHz, CDCl3) δ 1.33-

1.44 (m, 2H), 1.48-1.58 (m, 2H), 1.75-1.88 (m, 4H), 3.68 (brs, 2H), 3.91-4.01 (m, 7H), 6.74-6.79

(m, 1H), 6.90-6.96 (m, 3H), 6.98-7.03 (m, 2H), 7.08 (s, 1H), 7.44-7.51 (m, 3H); 13C NMR (75

MHz, CDCl3) δ 25.65, 26.33, 29.10, 31.03, 46.94, 55.54, 67.68, 109.13, 114.66, 115.15, 118.78,

119.35, 127.55, 129.39, 131.64, 134.21, 135.13, 137.06, 147.52, 157.85.

4'-(4-(1H-imidazol-1-yl)butoxy)-3-isopropoxy-[1,1'-biphenyl]-4-amine (3.32e). Yellow oil, 0.18

1 g, yield 90% starting from 0.22 g 3.31e (0.55 mmol); H NMR (400 MHz, CDCl3) δ 1.40 (d, J =

6.0 Hz, 6H), 1.76-1.86 (m, 2H), 1.98-2.07 (m, 2H), 3.56 (brs,2H), 3.99-4.08 (m, 4H), 4.63 (sep, J

= 6.0 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H) 6.88-6.95 (m, 2H), 6.96 (s, 1H), 6.97-7.02 (m, 2H), 7.10

13 (s, 1H), 7.44-7.48 (m, 2H), 7.54 (s, 1H); C NMR (100 MHz, CDCl3) δ 22.35, 26.34, 28.11,

178

46.80, 67.19, 70.86, 112.41, 114.63, 115.46, 118.81, 119.51, 127.59, 129.44, 131.42, 134.54,

136.41, 137.09, 145.58, 157.56.

4'-(6-((1H-imidazol-1-yl)hexyl)oxy)-3-isopropoxy-[1,1'-biphenyl]-4-amine (3.32f). Yellow oil,

1 0.23 g, yield 86% starting from 0.28 g 3.31f (0.66 mmol); H NMR (400 MHz, CDCl3) δ 1.34-

1.44 (m, 8H), 1.49-1.57 (m, 2H), 1.76-1.87 (m, 4H), 3.76 (brs, 2H), 3.93-4.00 (m, 4H), 4.62 (sep,

J = 6.0 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H) 6.90-6.96 (m, 3H), 6.97-7.04 (m, 2H), 7.08 (s, 1H),

13 7.43-7.47 (m, 2H), 7.49 (s, 1H); C NMR (100 MHz, CDCl3) δ 22.36, 25.65, 26.33, 29.11,

31.02, 46.94, 67.69, 70.85, 112.41, 114.67, 115.46, 118.78, 119.49, 127.52, 129.39, 131.52,

134.26, 136.36, 137.05, 145.57, 157.83.

4'-(4-(1H-imidazol-1-yl)butoxy)-2-methoxy-[1,1'-biphenyl]-4-amine (3.32g). Buff powder, 0.19

1 g, yield 83% starting from 0.25 g 3.31g (0.68 mmol); H NMR (300 MHz, CDCl3) δ 1.76-1.86

(m, 2H), 1.97-2.08 (m, 2H), 3.47 (brs, 2H), 3.78 (s, 3H), 3.99-4.09 (m, 4H), 6.32-6.38 (m, 2H),

6.88-6.92 (m, 2H), 6.97 (s, 1H), 7.08-7.12 (m, 2H), 7.40-7.46 (m, 2H), 7.58 (s, 1H); 13C NMR

(75 MHz, CDCl3) δ 26.31, 28.15, 46.88, 55.42, 67.06, 98.82, 107.36, 113.94, 118.86, 120.83,

129.16, 130.42, 131.31, 131.45, 137.00, 146.87, 157.21, 157.34.

4'-(6-((1H-imidazol-1-yl)hexyl)oxy)-2-methoxy-[1,1'-biphenyl]-4-amine (3.32h). Buff powder,

1 0.20 g, yield 94% starting from 0.23 g 3.31h (0.58 mmol); H NMR (400 MHz, CDCl3) δ 1.35-

1.44 (m, 2H), 1.49-1.58 (m, 2H), 1.76-1.88 (m, 4H), 3.60 (brs, 2H), 3.78 (s, 3H), 3.94-4.01 (m,

4H), 6.34 (d, J = 2.0 Hz, 1H), 6.36 (dd, J = 7.9, 2.0 Hz, 1H), 6.88-6.94 (m, 3H), 7.08-7.12 (m,

13 2H), 7.40-7.44 (m, 2H), 7.53 (s, 1H); C NMR (100 MHz, CDCl3) δ 25.65, 26.31, 29.11, 31.02,

179

47.03, 55.42, 67.54, 98.84, 107.37, 113.98, 118.83, 120.94, 129.19, 130.35, 131.15, 131.31,

136.99, 146.82, 157.34, 157.50.

4'-(4-(1H-imidazol-1-yl)butoxy)-2-isopropoxy-[1,1'-biphenyl]-4-amine (3.32i). Brown oil, 0.24

1 g, yield 99% starting from 0.26 g 3.31i (0.65 mmol); H NMR (300 MHz, CDCl3) δ 1.25 (d, J =

6.1 Hz, 6H), 1.78-1.86 (m, 2H), 1.98-2.07 (m, 2H), 3.56 (brs, 2H), 3.99-4.09 (m, 4H), 4.38 (sep,

J = 6.1 Hz, 1H), 6.33-6.38 (m, 2H), 6.86-6.91 (m, 2H), 6.97 (s, 1H), 7.08-7.13 (m, 2H), 7.42-

13 7.47 (m, 2H), 7.55 (s, 1H); C NMR (75 MHz, CDCl3) δ 22.11, 26.35, 28.16, 46.85, 67.03,

70.75, 102.52, 107.95, 113.71, 118.85, 122.21, 129.34, 130.37, 131.45, 131.87, 137.09, 146.61,

155.68, 157.04.

4'-(6-((1H-imidazol-1-yl)hexyl)oxy)-3-isopropoxy-[1,1'-biphenyl]-4-amine (3.32j). Yellow oil,

1 0.32 g, yield 98% starting from 0.35 g 3.31j (0.82 mmol); H NMR (300 MHz, CDCl3) δ 1.25 (d,

J = 6.1 Hz, 6H) 1.36-1.45 (m, 2H), 1.48-1.59 (m, 2H), 1.75-1.89 (m, 4H), 3.60 (brs, 2H), 3.93-

4.01 (m, 4H), 4.37 (sep, J = 6.1 Hz, 1H), 6.33-6.38 (m, 2H), 6.86-6.95 (m, 3H), 7.08 (s, 1H),

13 7.10 (d, J = 8.0 Hz, 1H), 7.42-7.47 (m, 2H), 7.49 (s, 1H); C NMR (75 MHz, CDCl3) δ 22.10,

25.66, 26.34, 29.15, 31.04, 46.93, 67.52, 70.77, 102.65, 107.98, 113.75, 118.76, 122.37, 129.44,

130.30, 131.44, 131.59, 137.08, 146.54, 155.69, 157.31.

180

Synthesis of biphenoxyalkyl AIA azole hybrids. (3.33a-j)

Compounds 3.33a-j were synthesized from 3.31a-j (0.38-0.60 mmol) following the general synthesis of compounds 3.12a-n.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-[1,1'-biphenyl]-4-yl)picolinimidamide (3.33a). Yellowish white powder, 130 mg, yield 65% starting from 150 mg 3.32a (0.48 mmol); mp 170-173°C; 1H

NMR (300 MHz, CDCl3) δ 1.77-1.84 (m, 2H), 1.99-2.09 (m, 2H), 4.01-4.09 (m, 4H), 5.91 (brs,

2H), 6.93-6.99 (m, 3H), 7.06-7.12 (m, 3H), 7.42 (ddd, J = 7.5, 4.9, 1.2 Hz, 1H), 7.51-7.60 (m,

5H), 7.85 (td, J = 7.9, 1.7 Hz, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.60 (ddd, J = 4.9, 1.7, 1.0 Hz, 1H);

+ HRMS (ESI) m/z (M +H) calcd for C25H26N5O, 412.21319; found, 412.21304; Anal. Calcd for

C25H25N5O: C, 72.97; H, 6.12; N, 17.02. Found: C, 72.95; H, 6.11; N, 16.85.

181

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-[1,1'-biphenyl]-4-yl)picolinimidamide (3.33b). Yellow powder, 107 mg, yield 60% starting from 137 mg 3.32b (0.40 mmol); mp 180-183°C; 1H NMR

(400 MHz, CDCl3) δ 1.38-1.44 (m, 2H), 1.52-1.58 (m, 2H), 1.77-1.88 (m, 4H), 3.96 (t, J = 7.1

Hz, 2H), 4.01 (6, J = 6.3Hz, 2H), 5.90 (brs, 2H), 6.93 (s, 1H), 6.95-6.99 (m, 2H), 7.04-7.14 (m,

3H), 7.42 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.48 (s, 1H), 7.52-7.60 (m, 4H), 7.84 (td, J = 7.7, 1.7

Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.60 (ddd, J = 4.8, 1.5, 0.9 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 25.65, 26.34, 29.11, 31.04, 46.93, 67.69, 114.76, 118.75, 121.62, 121.95, 125.16,

127.75 (overlapped 2Cs), 129.48, 133.56, 135.61, 136.80, 137.08, 147.87, 148.61, 151.57,

+ 152.68, 158.24; HRMS (ESI) m/z (M +H) calcd for C27H30N5O, 440.24449; found, 440.24475;

Anal. Calcd for C27H29N5O: C, 73.78; H, 6.65; N, 15.93. Found: C, 73.71; H, 6.76; N, 15.77.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-3-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide (3.33c).

Yellow powder, 108 mg, yield 59% starting from 140 mg 3.32c (0.41 mmol); mp 146-149 °C;

1 H NMR (400 MHz, CDCl3) δ 1.80-1.87 (m, 2H), 2.00-2.08 (m, 2H), 3.91 (s, 3H), 4.01-4.08 (m,

4H), 5.91 (brs, 2H), 6.95-6.99 (m, 3H), 7.07-7.14 (m, 2H), 7.15 (d, J = 1.7 Hz, 1H), 7.19 (dd, J =

7.9, 1.7 Hz, 1H), 7.41 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.51-7.58 (m, 3H), 7.84 (td, J = 7.7, 1.7

Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.60 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 26.35, 28.11, 46.76, 55.96, 67.21, 110.64, 114.70, 118.75, 119.77, 121.92, 122.99,

125.12, 127.92, 129.62, 134.06, 136.80, 136.85, 137.13, 147.83, 151.16, 151.40, 153.11, 158.07;

+ HRMS (ESI) m/z (M +H) calcd for C26H28N5O2, 442.22375; found, 442.22440; Anal. Calcd for

C26H27N5O2: C, 70.73; H, 6.16; N, 15.86. Found: C, 70.57; H, 6.33; N, 15.65.

182

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-3-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide

(3.33d). Yellow powder, 127 mg, yield 66% starting from 150 mg 3.32d (0.41 mmol); mp 150-

1 153 °C; H NMR (700 MHz, CDCl3) δ 1.38-1.43 (m, 2H), 1.52-1.57 (m, 2H), 1.79-1.87 (m,

4H), 3.90 (s, 3H), 3.97 (d, J = 7.1 Hz, 2H), 4.01 (d, J = 6.3 Hz, 2H), 5.90 (brs, 2H), 6.93 (t, d, J =

1.2 Hz, 1H), 6.96-6.99 (m, 2H), 7.04-7.15 (brs, 1H, overlapped), 7.08 (s, 1H), 7.15 (d, J = 1.8

Hz, 1H), 7.19 (dd, J = 7.9, 1.8 Hz, 1H), 7.41 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.49 (s, 1H), 7.53-

7.56 (m, 2H), 7.83 (td, J = 7.7, 1.7 Hz, 1H), 8.51 (d, J = 7.6 Hz, 1H), 8.59 (d, J = 4.6 Hz, 1H);

13 C NMR (100 MHz, CDCl3) δ 25.66, 26.35, 29.11, 31.05, 46.93, 55.95, 67.70, 110.59, 114.74,

118.76, 119.74, 121.90, 122.95, 125.10, 127.86, 129.50, 133.79, 136.79, 136.90, 137.10, 147.83,

+ 151.13, 151.48, 153.01, 158.33; HRMS (ESI) m/z (M +H) calcd for C28H32N5O2, 470.25505; found, 470.25528; Anal. Calcd for C28H31N5O2: C, 71.62; H, 6.65; N, 14.91. Found: C, 71.32; H,

6.82; N, 14.65.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-3-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide (3.33e).

Yellow powder, 70 mg, yield 36% starting from 150 mg 3.32e (0.41 mmol); mp 103-105 °C; 1H

NMR (400 MHz, CDCl3) δ 1.30 (d, J = 6.1 Hz, 6H), 1.80-1.87 (m, 2H), 2.00-2.09 (m, 2H), 4.01-

4.09 (m, 4H), 4.53 (sep, J = 6.1 Hz, 1H), 5.89 (brs, 2H), 6.93-6.98 (m, 3H), 7.02-7.14 (m, 2H),

7.20 (d, J = 1.8 Hz, 1H), 7.22 (dd, J = 7.9, 1.8 Hz, 1H), 7.41 (ddd, J = 7.5, 4.9, 1.1 Hz, 1H),

7.52-7.58 (m, 3H), 7.84 (td, J = 7.7, 1.7 Hz, 1H), 8.48 (m, 1H), 8.61 (d, J = 4.4 Hz, 1H); 13C

NMR (100 MHz, CDCl3) δ 22.38, 26.35, 28.12, 46.76, 67.20, 72.29, 114.69, 117.27, 118.75,

120.90, 121.75, 123.79, 124.99, 127.86, 129.63, 133.89, 136.52, 136.77, 137.13, 137.72, 147.87,

+ 149.20, 151.74, 152.19, 158.02; HRMS (ESI) m/z (M +H) calcd for C28H32N5O2, 470.25505;

183 found, 470.25504; Anal. Calcd for C28H31N5O2: C, 71.62; H, 6.65; N, 14.91. Found: C, 71.68; H,

6.74; N, 14.82.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-3-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide

(3.33f). Yellow powder, 78 mg, yield 41% starting from 150 mg 3.32f (0.38 mmol); mp 107-109

1 °C; H NMR (400 MHz, CDCl3) δ 1.30 (d, J = 6.1 Hz, 6H), 1.36-1.45 (m, 2H), 1.50-1.59 (m,

2H), 1.78-1.89 (m, 4H), 3.97 (t, J = 7.0 Hz, 2H), 4.01 (t, J = 6.3 Hz, 2H), 4.53 (sep, J = 6.1 Hz,

1H), 5.95 (brs, 2H), 6.93 (t, J = 1.1 Hz, 1H), 6.94-6.99 (m, 2H), 7.08 (t, J = 1.1 Hz, 1H), 7.10-

7.21 (m, 2H), 7.23 (dd, J = 7.09, 2.0 Hz, 1H), 7.42 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 7.49 (s, 1H),

7.51-7.56 (m, 2H), 7.85 (td, J = 7.7, 1.8 Hz, 1H), 8.48 (d, J = 7.9 Hz, 1H), 8.61 (ddd, J = 4.8,

13 1.6, 0.9 Hz, 1H); C NMR (100 MHz, CDCl3) δ 22.38, 25.65, 26.34, 29.10, 31.04, 46.93, 67.70,

72.28, 114.72, 116.95, 118.74, 120.83, 121.71, 123.56, 125.00, 127.80, 129.49, 133.59, 136.65,

136.78, 137.08, 147.88, 149.20, 151.70, 152.51, 158.29; HRMS (ESI) m/z (M +H)+ calcd for

C30H36N5O2, 498.28635; found, 498.28653; Anal. Calcd for C30H35N5O2: C, 72.41; H, 7.09; N,

14.07. Found: C, 72.46; H, 6.96; N, 13.91.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-2-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide (3.33g).

Yellow powder, 92 mg, yield 44% starting from 160 mg 3.32g (0.47 mmol); mp 129-131 °C; 1H

NMR (400 MHz, CDCl3) δ 1.81-1.85 (m, 2H), 1.99-2.08 (m, 2H), 3.82 (s, 3H), 4.02-4.08 (m,

4H), 5.92 (brs, 2H), 6.68-6.73 (m, 2H), 6.93-6.98 (m, 3H), 7.10 (s, 1H), 7.31 (d, J = 7.8 Hz,

1H), 7.41-7.46 (m, 1H), 7.49 (s, 1H), 7.51-7.54 (m, 2H), 7.85 (td, J = 7.8, 1.4 Hz, 1H), 8.46 (d, J

13 = 7.9 Hz, 1H), 8.61 (d, J = 4.6 Hz, 1H); C NMR (100 MHz, CDCl3) δ 26.34, 28.16, 46.75,

55.55, 67.08, 105.13, 113.44, 114.00, 118.76, 121.58, 124.98, 125.21, 129.60, 130.56, 131.20,

184

131.39, 136.82, 137.13, 147.91, 150.37, 151.52, 152.73, 157.44, 157.56; HRMS (ESI) m/z (M

+ +H) calcd for C26H28N5O2, 442.22375; found, 442.22458; Anal. Calcd for C26H27N5O2: C,

70.73; H, 6.16; N, 15.86. Found: C, 70.54; H, 6.13; N, 15.74.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide

(3.33h). Buff powder, 125 mg, yield 57% starting from 170 mg 3.32h (0.46 mmol); mp 102-104

1 °C; H NMR (400 MHz, CDCl3) δ 1.36-1.45 (m, 2H), 1.50-1.59 (m, 2H), 1.77-1.89 (m, 4H),

3.82 (s, 3H), 3.94-4.03 (m, 4H), 5.88 (brs, 2H), 6.68-6.72 (m, 2H), 6.92-6.97 (m, 3H), 7.08 (s,

1H), 7.31 (d, J = 8.0 Hz, 1H), 7.43 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 7.48-7.52 (m, 3H), 7.85 (td, J

= 7.8, 1.6 Hz, 1H), 8.47 (d, J = 8.0 Hz, 1H), 8.61 (d, J = 4.9 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 25.66, 26.33, 29.12, 31.05, 46.95, 55.56, 67.57, 105.17, 113.47, 114.04, 118.76,

121.64, 125.16, 125.24, 129.44, 130.50, 130.89, 131.40, 136.85, 137.07, 147.92, 149.97, 151.43,

+ 152.82, 157.45, 157.84; HRMS (ESI) m/z (M +H) calcd for C28H32N5O2, 470.25505; found,

470.25687; Anal. Calcd for C28H31N5O2: C, 71.62; H, 6.65; N, 14.91. Found: C, 71.46; H, 6.67;

N, 14.78.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-2-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide (3.33i).

Buff powder, 95 mg, yield 34% starting from 220 mg 3.32i (0.60 mmol); mp 99-101 °C; 1H

NMR (700 MHz, CDCl3) δ 1.28 (d, J = 6.0 Hz, 6H), 1.80-1.85 (m, 2H), 2.02-2.06 (m, 2H), 4.04

(d, J = 5.9 Hz, 2H), 4.07 (d, J = 7.1 Hz, 2H), 4.48 (sep, J = 6.0 Hz, 1H), 6.67-6.71 (m, 2H), 6.91-

6.94 (m, 2H), 6.97 (s, 1H), 7.10 (s, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.42 (ddd, J = 7.3, 4.8, 0.8 Hz,

1H), 7.51-7.54 (m, 3H), 7.85 (td, J = 7.7, 1.6 Hz, 1H), 8.45 (d, J = 7.9 Hz, 1H), 8.61 (d, J = 4.8

13 Hz, 1H); C NMR (175 MHz, CDCl3) δ 22.14, 26.37, 28.19, 46.78, 67.04, 70.69, 108.52,

185

113.76, 113.87, 118.78, 121.61, 125.21, 126.24, 129.60, 130.57, 131.60, 136.84, 137.15, 147.92,

+ 149.99, 151.54, 152.77, 155.75, 157.37; HRMS (ESI) m/z (M +H) calcd for C28H32N5O2,

470.25505; found, 470.25580; Anal. Calcd for C28H31N5O2: C, 71.62; H, 6.65; N, 14.91. Found:

C, 71.38; H, 6.65; N, 14.64.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide

(3.33j). Buff powder, 50 mg, yield 26% starting from 150 mg 3.32j (0.38 mmol); mp 120-123

1 °C; H NMR (400 MHz, CDCl3) δ 1.28 (d, J = 6.0 Hz, 6H), 1.37-1.45 (m, 2H), 1.51-1.59 (m,

2H), 1.78-1.89 (m, 4H), 3.95-4.03 (m, 4H), 4.47 (sep, J = 6.0 Hz, 1H), 6.01 (brs, 2H), 6.68-6.71

(m, 2H), 6.91-6.95 (m, 3H), 7.08 (s, 1H), 7.31-7.34 (m, 1H), 7.43 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H),

7.49-7.54 (m, 3H), 7.85 (td, J = 7.8, 1.7 Hz, 1H), 8.47 (d, J = 7.9 Hz, 1H), 8.61 (ddd, J = 4.3, 1.5,

13 0.9 Hz, 1H); C NMR (100 MHz, CDCl3) δ 22.13, 25.68, 26.35, 29.16, 31.05, 46.95, 67.53,

70.72, 108.63, 113.80, 113.92, 118.76, 121.66, 125.23, 126.47, 129.44, 130.50, 131.31, 131.59,

136.84, 137.07, 147.92, 149.60, 151.44, 152.84, 155.77, 157.65; HRMS (ESI) m/z (M +H)+ calcd for C30H36N5O2, 498.28635; found, 498.28766; Anal. Calcd for C30H35N5O2: C, 72.41; H,

7.09; N, 14.07. Found: C, 72.70; H, 7.05; N, 13.96.

186

Synthesis of 4-bromo-2-methoxyphenyl 4-methylbenzenesulfonate (3.35)

To a solution of 4-bromo-2-methoxyphenol (5 g, 24.62 mmol) in dry acetonitrile (50 mL) were added 1.5 equivalent of potassium carbonate (5.10 g, 36.94 mmol) and 1.5 equivalents of tosyl chloride (7.04 g, 36.94 mmol) and the mixture was refluxed overnight. After the reaction was complete, the suspension was filtered,the filtrate was evaporated under reduced pressure, and the crude product was purified by column chromatography using hexanes/DCM 1:1 as eluent

1 affording the pure product as a white solid, 7.7 g, yield 88%; H NMR (300 MHz, CDCl3) δ 2.46

(s, 3H), 3.56 (s, 3H), 6.96-6.98 (m, 1H), 7.01-7.05 (m, 2H), 7.29-7.35 (m, 2H), 7.72-7.78 (m,

13 2H); C NMR (75 MHz, CDCl3) δ 21.69, 55.82, 116.23, 120.84, 123.62, 125.21, 128.63,

129.44, 132.95, 137.57, 145.25, 152.49.

187

Synthesis of 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl 4-methylbenzenesulfonate (3.36)

A three necked flask was charged with 3.35 (7 g, 19.59 mmol), 1.3 equivalents bis(pinacolato)diboron (6.46 g, 25.47 mmol), 3 equivalents of potassium acetate (2.5 g, 25.47 mmol) and 6 mol% Pd(dppf)Cl2 (0.86 g). The flask was purged with N2 gas and degassed

o anhydrous dioxane was added (40 mL). The mixture was heated at 100 C under N2 atmosphere for 12 hours. After the reaction was complete, the suspension was cooled and distilled water (50 mL) and DCM (50 mL) were added. The aqueous layer was extracted twice with DCM (50 mL), the combined organic extract was dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography using hexanes/ethyl acetate 8:1 to 4:1 affording the product which was further crystallized from

1 methanol. White crystalline solid, 6.1 g, yield 77%; H NMR (400 MHz, CDCl3) δ 1.35 (s, 12H),

2.45 (s, 3H), 3.61 (s, 3H), 7.16 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 1.2 Hz, 1H), 7.28-7.32 (m, 2H),

188

13 7.36 (dd, J = 7.9, 1.2 Hz, 1H), 7.76 (m, 2H); C NMR (100 MHz, CDCl3) δ 21.65, 24.84, 55.57,

84.11, 118.32, 123.42, 127.46, 128.63, 129.31, 133.29, 140.81, 144.93, 151.18.

Synthesis of 3-methoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-yl 4-methylbenzenesulfonate (3.37a)

A three necked flask charged with 3.36 (3.13 g, 7.74 mmol), 1.2 equivalents of 4- nitroiodobenzene (2.29 g, 9.19 mmol), 2 equivalents of potassium carbonate (2.14 g, 15.48 mmol) and 6 mol % of Pd(dppf)Cl2 (0.34 g). Degassed DME/DMF/H2O (7:3:1 mL) were added

o and the mixture was heated at 100 C overnight under N2 atmosphere. After the reaction was complete, the liquid was concentrated under reduced pressure, distilled water (50 mL) was added, and the product was extracted with DCM (3 × 50 mL). The combined organic layer was dried over sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the crude product was purified by column chromatography using hexanes/DCM 1:1 to 1:2

1 affording 3.37a as yellow powder, 3 g, yield 97%; H NMR (300 MHz, CDCl3) δ 2.48 (s, 3H),

189

3.70 (s, 3H), 7.07 (d, J = 2.0 Hz, 1H), 7.15 (dd, J = 8.5, 2.0 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H),

7.33-7.38 (m, 2H), 7.68-7.72 (m, 2H), 7.81-7.85 (m, 2H), 8.29-8.33 (m, 2H); 13C NMR (100

MHz, CDCl3) δ 21.71, 55.82, 111.77, 119.79, 124.14, 124.62, 127.91, 128.60, 129.48, 133.30,

138.85, 139.03, 145.22, 146.57, 147.35, 152.33.

Synthesis of 3-isopropoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-yl 4-methylbenzenesulfonate (3.37b)

3.37a (0.65 g, 1.62 mmol) was dissolved in anhydrous DCM (5 mL) and cooled to -20 oC under

N2 atmosphere. Two equivalents of 1M BBr3 in DCM (3.3 mL, 3.3 mmol) was added dropwise and the mixture was further stirred for 4-5 hours at 0 oC. The reaction was quenched with water and the product was extracted with ethyl acetate (3 × 30 mL). The combined organic layer was dried over sodium sulfate and evaporated under reduced pressure. The crude product was purified by column chromatography using hexanes/DCM 6:1 to 4:1 as an eluent to afford the demethylated product as a yellow powder in 72% yield (0.45 g). The demethylated product (0.43

190 g, 1.11 mmol), 1.5 equivalents of potassium carbonate (0.23 g, 1.67 mmol) and 2 equivalents of isopropyl iodide (0.37 g, 2.23 mmol) were introduced into a sealed tube. Dry acetonitrile (5 mL) was added and the mixture was heated at 80 oC for 6 hr. After the reaction was complete, distilled water (20 mL) was added and the product was extracted with ethyl acetate (3 × 30 mL).

The combined organic layer was dried over sodium sulfate and filtered. The crude product was purified by column chromatography using hexanes/ethyl acetate 4:1 affording a yellow oil which upon addition of ethanol (5 mL) precipitated the pure product as a buff powder, 0.45 g, yield

1 95%; H NMR (300 MHz, CDCl3) δ 1.22 (d, J = 6.1 Hz, 6H), 2.48 (s, 3H), 4.53 (sep, J = 6.1 Hz,

1H), 7.07 (d, J = 2.0 Hz, 1H), 7.14 (dd, J = 8.4, 2.0 Hz, 1H), 7.31-7.38 (m, 3H), 7.66-7.70 (m,

13 2H), 7.81-7.85 (m, 2H), 8.28-8.32 (m, 2H); C NMR (100 MHz, CDCl3) δ 21.64, 21.68, 71.39,

114.43, 119.61, 124.12, 124.92, 127.89, 128.60, 129.57, 133.55, 138.61, 139.96, 145.13, 146.68,

147.30, 150.58.

191

Synthesis of biphenoxyalkyl AIA azole intermediates. (3.38a,b)

3.38a,b were synthesized according to a previously published procedure with minor modifications [169]. A 250 mL round bottom flask was charged with 3.37a,b (2.01-2.50 mmol).

Ethanol (80 mL), DMSO (20 mL), and 2N aqueous NaOH (20 mL) were added and the mixture was heated to reflux for 2-3 hr. After the reaction was complete, the solvent was removed under reduced pressure and the residue was acidified with 2N HCl solution, then the product was extracted with DCM (3 × 50 mL). The combined organic extract was dried over sodium sulfate and evaporated under reduced pressure to afford crude product which was purified by column chromatography to give 3.38a,b in 91-93% yield.

3-Methoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-ol. (3.38a). Chromatography solvent: hexanes/ethyl acetate

3:1 to 1:1, yellow powder, 0.57 g, yield 82% starting from 1.00 g 3.37a (2.50 mmol); 1H NMR

(400 MHz, CDCl3) δ 4.01 (s, 3H), 5.80 (s, 1H), 7.05 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 1.5 Hz,

192

1H), 7.19 (dd, J = 8.1, 1.5 Hz, 1H), 7.67-7.72 (m, 2H), 8.26-8.31 (m, 2H); 13C NMR (100 MHz,

CDCl3) δ 56.10, 109.66, 115.10, 120.85, 124.12, 127.21, 131.08, 146.60, 146.76, 147.05, 147.56.

3-Isopropoxy-4ʹ-nitro-[1,1ʹ-biphenyl]-4-ol. (3.38b). Chromatography solvent: hexanes/ethyl acetate 5:1 to 3:1, yellow powder, 0.50 g, yield 91% starting from 0.86 g 3.37b (2.01 mmol); 1H

NMR (400 MHz, CDCl3) δ 1.45 (d, J = 6.1 Hz, 6H), 4.72 (sep, J = 6.1 Hz, 1H), 5.88 (s, 1H),

7.06 (d, J = 8.2 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H) 7.79 (dd, J = 8.2, 2.0 Hz, 1H), 7.66-7.70 (m,

13 2H), 8.26-8.31 (m, 2H); C NMR (75 MHz, CDCl3) δ 22.19, 72.10, 112.32, 115.20, 120.82,

124.11, 127.19, 130.97, 145.11, 146.56, 147.63, 147.75.

Synthesis of biphenoxyalkyl AIA azole intermediates (3.39a-d)

Compounds 3.39a-d were synthesized from 3.38a,b (0.80-1.06 mmol) following the general synthesis of compounds 3.9a-i.

193

4-(Bromobutoxy)-3-methoxy-4ʹ-nitro-1,1ʹ-biphenyl (3.39a). Buff powder, 0.38 g, yield 94%

1 starting from 0.26 g 3.38a (1.06 mmol); H NMR (300 MHz, CDCl3) δ 1.99-2.18 (m, 4H), 3.54

(t, J = 6.4 Hz, 2H), 3.96 (s, 3H), 4.13 (d, J = 6.1 Hz, 2H), 6.99 (d, J = 8.3 Hz, 1H), 7.12 (d, J =

2.1 Hz, 1H), 7.20 (dd, J = 8.3, 2.1 Hz, 1H), 7.68-7.74 (m, 2H), 8.26-8.31 (m, 2H); 13C NMR (75

MHz, CDCl3) δ 27.81, 29.45, 33.35, 56.20, 68.16, 110.98, 113.40, 120.14, 124.11, 127.25,

131.78, 146.67, 147.38, 149.39, 149.96.

4-(Bromobutoxy)-3-isopropoxy-4ʹ-nitro-1,1ʹ-biphenyl (3.39b). Yellow powder, 0.30 g, yield

1 91% starting from 0.22 g 3.38b (0.80 mmol); H NMR (300 MHz, CDCl3) δ 1.40 (d, J = 6.0 Hz,

6H), 1.97-2.19 (m, 4H), 3.56 (t, J = 6.5 Hz, 2H), 4.11 (t, J = 6.0 Hz, 2H), 4.58 (sep, J = 6.0 Hz,

1H), 6.99 (d, J = 8.3 Hz, 1H), 7.19-7.24 (m, 2H), 7.66-7.71 (m, 2H), 8.24-8.30 (m, 2H); 13C

NMR (75 MHz, CDCl3) δ 22.26, 27.85, 29.69, 33.48, 68.28, 72.57, 114.34, 116.88, 121.00,

124.11, 127.20, 131.79, 146.63, 147.28, 148.18, 151.20.

4-((6-Bromohexyl)oxy)-3-methoxy-4'-nitro-1,1'-biphenyl (3.39c). Yellow powder, 0.36 g, yield

1 83% starting from 0.26 g 3.38a (1.06 mmol); H NMR (300 MHz, CDCl3) δ 1.52-1.59 (m, 4H),

1.86-1.97 (m, 4H), 3.44 (t, J = 6.9 Hz, 2H), 3.97 (s, 3H), 4.10 (t, J = 6.6 Hz, 2H), 6.97 (d, J =

8.3 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 7.20 (dd, J = 8.3, 2.1 Hz, 1H), 7.69-7.73 (m, 2H), 8.26-

13 8.31 (m, 2H); C NMR (75 MHz, CDCl3) δ 25.22, 27.90, 28.94, 32.64, 33.72, 56.21, 68.91,

110.92, 113.20, 120.14, 124.10, 127.22, 131.49, 146.63, 147.43, 149.58, 149.89.

4-((6-Bromohexyl)oxy)-3-isopropoxy-4'-nitro-1,1'-biphenyl (3.39d). Yellow powder, 0.32 g,

1 yield 93% starting from 0.22 g 3.38b (0.80 mmol); H NMR (400 MHz, CDCl3) δ 1.40 (d, J =

6.0 Hz, 6H), 1.54-1.60 (m, 4H), 1.86-1.97 (m, 4H), 3.46 (t, J = 6.7 Hz, 2H), 4.08 (t, J = 6.4 Hz,

194

2H), 4.57 (sep, J = 6.0 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 7.21-7.26 (m, 2H), 7.67-7.72 (m, 2H),

13 8.26-8.30 (m, 2H); C NMR (75 MHz, CDCl3) δ 22.29, 25.27, 27.87, 29.05, 32.68, 33.72, 68.88,

72.85, 114.20, 117.48, 121.20, 124.10, 127.16, 131.47, 146.58, 147.29, 148.11, 151.57.

Synthesis of biphenoxyalkyl AIA azole intermediates. (3.40a-d)

Compounds 3.40a-d were synthesized from 3.39a-d (0.68-0.84 mmol) following the general synthesis of compounds 3.10a-m.

1-(4-((3-Methoxy-4ʹ-nitro-[1,1ʹ-biphenyl])-4-yl)oxy)butyl)-1H-imidazole. (3.40a) Yellow

1 powder, 0.25 g, yield 81% starting from 0.32 g 3.39a (0.84 mmol) H NMR (300 MHz, CDCl3) δ

1.81-1.91 (m, 2H), 1.99-2.10 (m, 2H), 3.95 (s, 3H), 4.06-4.13 (m, 4H), 6.92-6.99 (m, 2H), 7.07

(s, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.18 (dd, J = 8.3, 2.0 Hz, 1H), 7.55 (s, 1H), 7.67-7.72 (m, 2H),

13 8.24-8.29 (m, 2H); C NMR (75 MHz, CDCl3) δ 25.97, 28.32, 46.65, 50.07, 68.58, 110.80,

113.24, 118.78, 120.10, 124.10, 127.26, 129.53, 131.85, 137.25, 146.67, 147.33, 149.24, 149.89.

195

1-(4-((3-Isopropoxy-4ʹ-nitro-[1,1ʹ-biphenyl])-4-yl)oxy)butyl)-1H-imidazole. (3.40b) Yellow

1 powder, 0.21 g, yield 77% starting from 0.28 g 3.39b (0.68 mmol) H NMR (400 MHz, CDCl3) δ

1.40 (d, J = 6.0 Hz, 6H), 1.82-1.89 (m, 2H), 2.01-2.10 (m, 2H), 4.09 (t, J = 5.9 Hz, 2H), 4.14 (t, J

= 6.7 Hz, 2H), 4.60 (d, J = 6.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H),7.00 (s, 1H), 7.10 (s, 1H), 7.18-

13 7.24 (m, 2H), 7.58 (s, 1H), 7.67-7.71 (m, 2H), 8.27-8.31 (m, 2H); C NMR (100 MHz, CDCl3) δ

22.24, 25.97, 28.51, 46.67, 68.70, 72.16, 114.09, 115.97, 118.81, 120.73, 124.12, 127.23, 129.55,

131.89, 137.29, 146.66, 147.28, 148.12, 150.84.

1-(6-((3-Methoxy-4ʹ-nitro-[1,1ʹ-biphenyl])-4-yl)oxy)hexyl)-1H-imidazole. (3.40c) Yellow

1 powder, 0.23 g, yield 74% starting from 0.32 g 3.39c (0.78 mmol) H NMR (300 MHz, CDCl3) δ

1.34-1.46 (m, 2H), 1.49-1.60 (m, 2H), 1.78-1.93 (m, 4H), 3.96 (s, 3H), 3.92-3.99 (m, 2H), 4.07

(t, J = 6.5 Hz, 2H), 6.92 (s, 1H), 6.96 (d, J = 8.3 Hz, 1H), 7.07 (s, 1H), 7.13 (d, J = 2.0 Hz, 1H),

7.19 (dd, J = 8.3, 2.0 Hz, 1H), 7.47 (s, 1H), 7.67-7.74 (m, 2H), 8.25-8.30 (m, 2H); 13C NMR (75

MHz, CDCl3) δ 25.51, 26.27, 28.91, 30.96, 46.86, 56.18, 68.76, 110.89, 113.17, 118.73, 120.13,

124.09, 127.22, 129.47, 131.54, 137.06, 146.64, 147.40, 149.49, 149.86.

1-(6-((3-Isopropoxy-4ʹ-nitro-[1,1ʹ-biphenyl])-4-yl)oxy)hexyl)-1H-imidazole. (3.40d) Yellow

1 powder, 0.25 g, yield 88% starting from 0.30 g 3.39d (0.68 mmol) H NMR (400 MHz, CDCl3) δ

1.37-1.45 (m, 8H), 1.52-1.60 (m, 2H), 1.80-1.90 (m, 4H), 3.97 (t, J = 7.0 Hz, 2H), 4.05 (t, J = 6.3

Hz, 2H), 4.56 (d, J = 6.0 Hz, 1H), 6.92 (s, 1H), 6.98 (d, J = 8.1 Hz, 1H), 7.07 (s, 1H), 7.20-7.25

13 (m, 2H), 7.48 (s, 1H), 7.67-7.71 (m, 2H), 8.26-8.30 (m, 2H); C NMR (100 MHz, CDCl3) δ

22.29, 25.56, 26.26, 29.03, 31.03, 46.89, 68.78, 72.76, 114.22, 117.32, 118.75, 121.16, 124.10,

127.16, 129.47, 131.55, 137.07, 146.59, 147.27, 148.11, 151.44.

196

Synthesis of biphenoxyalkyl AIA azole intermediates (3.41a-d)

Compounds 3.41a-d were synthesized from 3.40a-d (0.48-0.54 mmol) following the general synthesis of compounds 3.11a-m.

4ʹ-(4-(1H-imidazol-1-yl)butoxy)-3ʹ-methoxy-[1,1ʹ-biphenyl]-4-amine (3.41a). Yellow powder,

1 0.17 g, yield 93% starting from 0.20 g 3.40a (0.54 mmol); H NMR (400 MHz, CDCl3) δ 1.81-

1.89 (m, 2H), 1.99-2.07 (m, 2H), 3.70 (brs, 2H), 3.93 (s, 3H), 4.05-4.12 (m, 4H), 6.74-6.78 (m,

2H), 6.90 (d, J = 8.5 Hz, 1H), 6.98 (s, 1H), 7.05-7.10 (m, 3H), 7.36-7.40 (m, 2H), 7.56 (s, 1H);

13 C NMR (100 MHz, CDCl3) δ 26.10, 28.38, 46.71, 55.92, 68.66, 110.41, 113.59, 115.37,

118.59, 118.82, 127.31, 129.46, 131.40, 134.94, 137.25, 145.57, 147.05, 149.62.

4ʹ-(4-(1H-imidazol-1-yl)butoxy)-3ʹ-isopropoxy-[1,1ʹ-biphenyl]-4-amine (3.41b). Buff powder,

1 0.16 g, yield 91% starting from 0.19 g 3.40b (0.48 mmol); H NMR (400 MHz, CDCl3) δ 1.37

(d, J = 6.0 Hz, 6H), 1.78-1.87 (m, 2H), 2.00-2.08 (m, 2H), 3.37 (brs, 2H), 4.06 (t, J = 5.8 Hz,

197

2H), 4.13 (t, J = 7.1 Hz, 2H), 4.57 (sep, J = 6.0 Hz, 1H), 6.73-6.79 (m, 2H), 6.91 (d, J = 8.3 Hz,

1H), 7.00 (s, 1H), 7.06-7.12 (m, 3H), 7.34-7.39 (m, 2H), 7.58 (s, 1H); 13C NMR (100 MHz,

CDCl3) δ 22.31, 26.03, 28.59, 46.81, 68.88, 71.76, 114.51, 115.34, 115.37, 118.97, 119.31,

127.70, 129.09, 131.37, 134.96, 137.20, 145.49, 147.88, 148.59.

4ʹ-((6-(1H-imidazol-1-yl)hexyl)oxy)-3ʹ-methoxy-[1,1ʹ-biphenyl]-4-amine (3.41c). yellow

1 powder, 0.16 g, yield 89% starting from 0.20 g 3.40c (0.50 mmol); H NMR (300 MHz, CDCl3)

δ 1.34-1.45 (m, 2H), 1.48-1.60 (m, 2H), 1.77-1.99 (m, 4H), 3.72 (brs, 2H), 3.92 (s, 3H), 3.90-

3.99 (m, 2H, overlapped), 4.04 (t, J = 6.6 Hz, 2H), 6.73-6.78 (m, 2H), 6.88-6.93 (m, 2H), 7.03-

13 7.09 (m, 3H), 7.35-7.40 (m, 2H), 7.47 (s, 1H); C NMR (75 MHz, CDCl3) δ 25.53, 26.30, 29.03,

30.98, 46.90, 56.02, 68.85, 110.50, 113.49, 115.37, 118.61, 118.74, 127.70, 129.43, 131.51,

134.59, 137.05, 145.47, 147.29, 149.57.

4ʹ-((6-(1H-imidazol-1-yl)hexyl)oxy)-3ʹ-isopropoxy-[1,1ʹ-biphenyl]-4-amine (3.41d). orange oil,

1 0.18 g, yield 88% starting from 0.22 g 3.40d (0.52 mmol); H NMR (400 MHz, CDCl3) δ 1.33-

1.44 (m, 8H), 1.50-1.60 (m, 2H), 1.78-1.88 (m, 4H), 3.12 (brs, 2H), 3.96 (t, J = 7.1 Hz, 2H), 4.01

(t, J = 6.4 Hz, 2H), 4.53 (sep, J = 6.0 Hz, 1H), 6.73-6.77 (m, 2H), 6.89-6.93 (m, 2H), 7.05-7.13

13 (m, 3H), 7.34-7.38 (m, 2H), 7.49 (s, 1H); C NMR (100 MHz, CDCl3) δ 22.35, 25.59, 26.29,

29.16, 31.04, 46.93, 69.00, 72.40, 114.69, 115.38, 116.66, 118.79, 119.72, 127.64, 129.35,

131.36, 134.63, 137.05, 145.44, 147.91, 149.19.

198

Synthesis of biphenoxyalkyl AIA azole analogs (3.42a-d)

Compounds 3.42a-d were synthesized from 3.41a-d (0.35-0.44 mmol) following the general synthesis of compounds 3.12a-n.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-3'-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide (3.42a).

Buff powder, 118 mg, yield 60% starting from 150 mg 3.41a (0.44 mmol); mp 160-162 °C; 1H

NMR (400 MHz, CDCl3) δ 1.83-1.91 (m, 2H), 2.01-2.09 (m, 2H), 3.96 (s, 3H), 4.08-4.14 (m,

4H), 5.96 (brs, 2H), 6.92-6.96 (m, 1H), 6.99 (s, 1H), 7.08-7.13 (m, 3H), 7.14-7.19 (m, 2H), 7.43

(ddd, J = 7.5, 4.9, 1.1 Hz, 1H), 7.56 (s, 1H), 7.57-7.61 (m, 2H), 7.85 (td, J = 7.7, 1.7 Hz, 1H),

13 8.46 (d, J = 7.9 Hz, 1H), 8.61 (d, J = 4.9 Hz, 1H); C NMR (100 MHz, CDCl3) δ 26.09, 28.39,

46.71, 55.96, 68.64, 110.61, 113.51, 118.80, 119.01, 121.61, 121.96, 125.19, 127.91, 129.51,

134.62, 135.73, 136.81, 137.27, 147.49, 147.89, 149.05, 149.67, 151.53, 152.67; HRMS (ESI)

199

+ m/z (M +H) calcd for C26H28N5O2, 442.22375; found, 442.22209; Anal. Calcd for C26H27N5O2:

C, 70.73; H, 6.16; N, 15.86. Found: C, 70.58; H, 6.32; N, 15.88.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-3'-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide

(3.42b). Yellow powder, 95 mg, yield 53% starting from 140 mg 3.41b (0.38 mmol); mp 133-

1 135 °C; H NMR (700 MHz, CDCl3) δ 1.39 (d, J = 6.1 Hz, 6H), 1.81-1.86 (m, 2H), 2.03-2.07

(m, 2H), 4.08 (t, J = 5.9 Hz, 2H), 4.13 (d, J = 7.1 Hz, 2H), 4.60 (sep, J = 6.1 Hz, 1H), 5.98 (brs,

2H), 6.94 (d, J = 8.3 Hz, 1H), 7.00 (s, 1H), 7.08-7.11 (m, 3H), 7.16 (dd, J = 8.3, 2.0 Hz, 1H),

7.19 (d, J = 2.0 Hz, 1H), 7.41-7.43 (m, 1H), 7.56-7.59 (m, 3H), 7.85 (td, J = 7.8, 1.3 Hz, 1H),

13 8.45 (d, J = 7.8 Hz, 1H), 8.60 (d, J = 4.8 Hz, 1H); C NMR (175 MHz, CDCl3) δ 22.33, 26.07,

28.59, 46.72, 68.82, 71.85, 114.41, 115.61, 118.87, 119.71, 121.63, 121.96, 125.21, 128.88,

129.48, 134.61, 135.67, 136.83, 137.31, 147.90, 147.94, 148.83, 149.07, 151.52, 152.71; HRMS

+ (ESI) m/z (M +H) calcd for C28H32N5O2, 470.25505; found, 470.25583; Anal. Calcd for

C28H31N5O2: C, 71.62; H, 6.65; N, 14.91. Found: C, 71.64; H, 6.64; N, 14.97.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-3'-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide

(3.42c). Yellow powder, 70 mg, yield 39% starting from 140 mg 3.41c (0.38 mmol); mp 145-147

1 °C; H NMR (400 MHz, CDCl3) δ 1.37-1.46 (m, 2H), 1.51-1.60 (m, 2H), 1.80-1.92 (m, 4H),

3.96 (s, 3H), 3.94-3.99 (m, 2H, overlapped), 4.07 (t, J = 6.5 Hz, 2H), 5.85 (brs, 2H), 6.92-6.97

(m, 2H), 7.08 (s, 1H), 7.09-7.13 (m, 2H), 7.14-7.18 (m, 2H), 7.43 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H),

7.49 (s, 1H), 7.57-7.61 (m, 2H), 7.85 (td, J = 7.8, 1.7 Hz, 1H), 8.47 (d, J = 8.0 Hz, 1H), 8.61 (d, J

13 = 4.3 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.55, 26.32, 29.02, 31.00, 46.92, 56.06, 68.81,

110.66, 113.38, 118.76, 119.02, 121.65, 121.98, 125.22, 127.89, 129.42, 134.24, 135.86, 136.83,

200

137.06, 147.73, 147.90, 148.62, 149.61, 151.45, 152.76; HRMS (ESI) m/z (M +H)+ calcd for

C28H32N5O2, 470.25505; found, 470.25346; Anal. Calcd for C28H31N5O2: C, 71.62; H, 6.65; N,

14.91. Found: C, 71.35; H, 6.87; N, 14.64.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-3'-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide

(3.42d). Yellowish white powder, 85 mg, yield 48% starting from 140 mg 3.41d (0.35 mmol);

1 mp 129-131 °C; H NMR (400 MHz, CDCl3) δ 1.36-1.46 (m, 8H), 1.52-1.61 (m, 2H), 1.80-1.89

(m, 4H), 3.97 (t, J = 7.2 Hz, 2H), 4.04 (t, J = 6.4 Hz, 2H), 4.56 (sep, J = 6.0 Hz, 1H), 5.99 (brs,

2H), 6.93 (s, 1H), 6.96 (d, J = 8.2 Hz, 1H), 7.07-7.12 (m, 3H), 7.17-7.21 (m, 2H), 7.43 (ddd, J =

7.5, 4.8, 1.1 Hz, 1H), 7.49 (s, 1H), 7.56-7.60 (m, 2H), 7.85 (td, J = 7.8, 1.7 Hz, 1H), 8.47 (d, J =

13 7.9 Hz, 1H), 8.61 (ddd, J = 4.8 Hz, 1H) ; C NMR (100 MHz, CDCl3) δ 22.36, 25.60, 26.30,

29.16, 31.06, 46.92, 68.96, 72.46, 114.61, 116.90, 118.76, 120.12, 121.64, 121.94, 125.19,

127.83, 129.46, 134.30, 135.69, 136.82, 137.07, 147.88, 147.97, 148.65, 149.66, 151.52, 152.72;

+ HRMS (ESI) m/z (M +H) calcd for C30H36N5O2, 498.28635; found, 498.28506; Anal. Calcd for

C30H35N5O2: C, 72.41; H, 7.09; N, 14.07. Found: C, 72.36; H, 7.10; N, 13.92.

201

Synthesis of 4-bromo-3-methoxyphenyl 4-methylbenzenesulfonate. (3.44)

3.44 was synthesized according to previously published procedure[170]. To a solution of 4- bromobenzene-1,3-diol 2.45 g, 12.96 mmol) in acetone was added 3 equivalents of potassium carbonate (5.38 g, 38.94 mmol) and 1.1 equivalent of tosyl chloride (2.73 g, 14.3 mmol) and the mixture was refluxed for 21h. Three equivalents of CH3I (5.00 g, 35.2 mmol) was added the mixture was further refluxed for 3h. After reaction completeion the suspension was filtered and the filtrate was concentrated under reduced pressure. Distilled water (100 mL) was added and the product was extracted with ethyl acetate (3 × 50 mL). The combined organic layer was washed with water and brine, dried over sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and the product was purified by column chromatography using hexanes/ethyl

1 acetate 3:1 to afford 3.44 as a white powder, 3.75 g, yield 81%. H NMR (400 MHz, CDCl3) δ

2.47 (s, 3H), 3.80 (s, 3H), 6.42 (dd, J = 8.7, 2.5 Hz, 1H), 6.61 (d, J = 2.5 Hz, 1H), 7.33-7.36 (m,

2H), 7.42 (d, J = 8.7 Hz, 1H), 7.72-7.75 (m, 2H).

202

Synthesis of 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl 4- methylbenzenesulfonate. (3.45)

Compound 3.45 was synthesized from 3.44 (3.70 g, 10.35 mmol) following the synthetic procedure of 3.37. Chromatography solvent: hexanes/ethyl acetate 6:1. White crystalline powder,

1 1.85 g, yield 44%; H NMR (400 MHz, CDCl3) δ 1.34 (s, 12H), 2.45 (s, 3H), 3.71 (s, 3H), 6.50-

6.54 (m, 2H), 7.29-7.33 (m, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.69-7.73 (m, 2H); 13C NMR (100

MHz, CDCl3) δ 21.67, 24.78, 55.85, 83.69, 105.16, 113.65, 128.62, 129.69, 132.31, 137.42,

145.33, 152.96, 165.05.

203

Synthesis of biphenoxyalkyl AIA azole hybid intermediates. (3.46a,c)

A three necked flask was charged with 3.45 (3.46-4.94 mmol), 1 equivalent of 4- nitroiodobenzene (0.86 g, 3.46 mmol) or 1 equivalent of 1-bromo-2-methoxy-4-nitrobenzene

(1.14 g, 4.94 mmol), 3 equivalents of potassium carbonate (1.43-2.05 g, 10.38-14.84 mmol) and

8 mol % of Pd(dppf)Cl2 (0.20-0.28 g). Degassed DME:DMF:H2O (7:3:1 mL) were added and

o the mixture was heated at 100 C overnight under N2 atmosphere. After the reaction was complete, the liquid was concentrated under reduced pressure, distilled water (50 mL) was added, and the product was extracted with DCM (3 × 50 mL). The combined organic layer was dried over sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the crude product was purified by column chromatography affording the product in 69-85% yield.

204

2-Methoxy-4'-nitro-[1,1'-biphenyl]-4-yl 4-methylbenzenesulfonate. (3.46a) Chromatography solvent: hexanes/DCM 3:4. Yellow powder, 1.17 g, yield 85% starting from 1.40 g of 3.45 (3.46

1 mmol); H NMR (300 MHz, CDCl3) δ 2.49 (s, 3H), 3.75 (s, 3H), 6.65 (dd, J = 8.3, 2.2 Hz, 1H),

6.74 (d, J = 2.2 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.35-7.40 (m, 2H), 7.62-7.66 (m, 2H), 7.79-

13 7.84 (m, 2H), 8.23-8.28 (m, 2H); C NMR (75 MHz, CDCl3) δ 21.73, 55.82, 106.41, 114.48,

123.27, 127.16, 128.58, 129.84, 130.26, 131.05, 132.50, 144.10, 145.59, 146.89, 150.73, 157.11.

2,2'-Dimethoxy-4'-nitro-[1,1'-biphenyl]-4-yl 4-methylbenzenesulfonate. (3.46c) Chromatography solvent: hexanes/DCM 1:1 to 1:2. Yellow powder, 1.41 g, yield 67% starting from 2.00 g of 3.45

1 (4.94 mmol); H NMR (400 MHz, CDCl3) δ 2.49 (s, 3H), 3.69 (s, 3H), 3.87 (s, 3H), 6.62 (dd, J =

8.3, 2.2 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H),

7.36-7.40 (m, 2H), 7.80-7.84 (m, 3H), 7.88 (dd, J = 8.3, 2.2 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 21.73, 55.82, 56.10, 105.96, 106.09, 113.84, 115.53, 124.47, 128.58, 129.80, 131.36,

131.81, 132.65, 133.58, 145.48, 148.38, 150.46, 157.41, 157.49.

205

Synthesis of biphenoxyalkyl AIA azole hybid intermediates (3.46b,d)

Compounds 3.46b,d were synthesized from 3.46a (1.00g, 2.5 mmol) or 3.46c (0.92 g, 2.14 mmol) following the synthetic procedure of 3.37b except that 4 equivalents of 1M BBr3 in DCM

(8.56 mmol) was used in the synthesis of 3.46d.

2-Isopropoxy-4'-nitro-[1,1'-biphenyl]-4-yl 4-methylbenzenesulfonate (3.46b) First step:

Chromatography solvent: hexanes/ethyl acetate 3:1 followed by washing the solid product with hexanes/diethyl ether 1:2. Buff powder, 0.45 g, yield 47% starting from 1 g 3.46a (2.50 mmol).

Second step: Chromatography solvent: hexanes/ethyl acetate 5:1. Yellow oil, 0.85 g, yield 89%

1 starting from 0.86 g demethylated starting material (2.23 mmol); H NMR (400 MHz, CDCl3) δ

1.23 (d, J = 6.0 Hz, 6H), 2.48 (s, 3H), 4.42 (sep, J = 6.0 Hz, 1H), 6.63 (dd, J = 8.4, 2.1 Hz, 1H),

6.69 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.35-7.39 (m, 2H), 7.63-7.68 (m, 2H), 7.78-

13 7.83 (m, 2H), 8.23-8.28 (m, 2H); C NMR (100 MHz, CDCl3) δ 21.67, 21.70, 71.24, 108.69,

206

114.36, 123.15, 127.97, 128.57, 129.85, 130.25, 131.24, 132.47, 144.49, 145.54, 146.76, 150.56,

155.40.

2,2'-Diisopropoxy-4'-nitro-[1,1'-biphenyl]-4-yl 4-methylbenzenesulfonate (3.46d) First step:

Chromatography solvent: DCM/ethyl acetate 10:1. Brown oil, 0.55 g, yield 64% starting from

0.92 g 3.46c (2.14 mmol). Second step: Chromatography solvent: hexanes/ethyl acetate 4:1. Buff powder from hexanes, 0.37 g, yield 62% starting from 0.5 g demethylated starting material (1.24

1 mmol); H NMR (400 MHz, CDCl3) δ 1.14 (d, J = 6.0 Hz, 6H), 1.24 (d, J = 6.0 Hz, 6H), 2.47 (s,

3H), 4.31 (sep, J = 6.0 Hz, 1H), 4.52 (sep, J = 6.0 Hz, 1H), 6.58 (dd, J = 8.4, 2.1 Hz, 1H), 6.63

(d, J = 2.1 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.35-7.39 (m, 3H), 7.76-7.80 (m, 3H), 7.83 (d, J =

13 2.0 Hz, 1H); C NMR (100 MHz, CDCl3) δ 21.67 (overlapped 2Cs), 21.75, 70.99, 71.60,

108.33, 108.71, 113.50, 115.12, 125.91, 128.62, 129.72, 131.63, 132.06, 132.47, 135.32, 145.37,

148.08, 150.08, 155.74, 155.75.

207

Synthesis of biphenoxyalkyl AIA azole hybid intermediates (3.47a-d)

Compounds 3.47a-d were synthesized from 3.46a-d (0.74-2.75 mmol) following the general synthesis of compounds 3.38a,b except that ethanol was used as a solvent instead of ethanol and

DMSO in the preparation of compounds 3.47c,d.

2-Methoxy-4'-nitro-[1,1'-biphenyl]-4-ol (3.47a). Chromatography solvent: hexanes/ethyl acetate

4:1 to 1:1. Yellow powder, 0.61 g, yield 91% starting from 1.1 g 3.46a (2.75 mmol); 1H NMR

(400 MHz, CDCl3) δ 3.84 (s, 3H), 5.11 (s, 1H), 6.53-6.58 (m, 2H), 7.23 (d, J = 8.2 Hz, 1H),

13 7.65-7.70 (m, 2H), 8.23-8.27 (m, 2H); C NMR (100 MHz, CDCl3) δ 55.62, 99.55, 107.73,

121.05, 123.26, 130.02, 131.52, 145.34, 146.23, 157.51, 157.82.

2-Isopropoxy-4'-nitro-[1,1'-biphenyl]-4-ol. (3.47b) Chromatography solvent: hexanes/ethyl acetate 3:1. Buff powder from hexanes, 0.50 g, yield 92% starting from 0.85 g of 3.46b (1.98

1 mmol); H NMR (400 MHz, CDCl3) δ 1.31 (d, J = 6.0 Hz, 6H), 4.53 (sep, J = 6.0 Hz, 1H), 4.96

208

(s, 1H), 6.50-6.56 (m, 2H), 7.23 (d, J = 8.4 Hz, 1H), 7.67-7.71 (m, 2H), 8.22-8.26 (m, 2H); 13C

NMR (75 MHz, CDCl3) δ 21.94, 70.97, 101.92, 107.78, 122.08, 123.11, 130.01, 131.68, 145.69,

146.13, 156.16, 157.26.

2,2'-Dimethoxy-4'-nitro-[1,1'-biphenyl]-4-ol. (3.47c) Chromatography solvent: hexanes/DCM

1:2. Yellow powder from hexane/diethyl ether 2:1, 0.25 g, yield 87% starting from 0.45 g of

1 3.46c (1.04 mmol); H NMR (400 MHz, CDCl3) δ 3.77 (s, 3H), 3.89 (s, 3H), 5.28 (s, 1H), 6.50

(dd, J = 8.2, 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 8.3 Hz,

13 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.89 (dd, J = 8.3, 2.2 Hz, 1H); C NMR (100 MHz, CDCl3) δ

55.67, 56.12, 99.43, 105.94, 107.10, 115.61, 118.03, 131.69, 132.04, 134.80, 147.88, 157.20,

157.52, 157.99.

2,2'-Diisopropoxy-4'-nitro-[1,1'-biphenyl]-4-ol. (3.47d) Chromatography solvent: hexanes/ethyl acetate 3:1. Orange oil, 0.23 g, yield 94% starting from 0.36 g of 3.46d (0.74 mmol); 1H NMR

(400 MHz, CDCl3) δ 1.22 (d, J = 6.0 Hz, 6H), 1.28 (d, J = 6.0 Hz, 6H), 4.41 (sep, J = 6.0 Hz,

1H), 4.54 (sep, J = 6.0 Hz, 1H), 4.93 (s, 1H), 6.46 (dd, J = 8.3, 2.3 Hz, 1H), 6.51 (d, J = 2.3 Hz,

1H), 7.11 (d, J = 8.3 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.83 (dd, J =

13 8.4, 2.2 Hz, 1H); C NMR (100 MHz, CDCl3) δ 21.81, 21.96, 70.87, 71.50, 101.88, 106.88,

108.88, 115.12, 119.78, 132.05, 132.48, 136.45, 147.54, 155.85, 156.34, 156.55.

209

Synthesis of biphenoxyalkyl AIA azole hybid intermediates (3.48a-f)

Compounds 3.48a-f were synthesized from 3.47a-d (0.63-1.22 mmol) following the general synthesis of compounds 3.9a-i.

4-(4-Bromobutoxy)-2-methoxy-4'-nitro-1,1'-biphenyl. (3.48a) Yellow powder, 0.40 g, yield 86%

1 starting from 0.30 g 3.47a (1.22 mmol); H NMR (400 MHz, CDCl3) δ 1.97-2.05 (m, 2H), 2.09-

2.16 (m, 2H), 3.53 (t, J = 6.5 Hz, 2H), 3.85 (s, 3H), 4.08 (t, J = 6.1 Hz, 2H), 6.58-6.62 (m, 2H),

13 7.26-7.30 (m, 1H), 7.66-7.70 (m, 2H), 8.23-8.27 (m, 2H); C NMR (100 MHz, CDCl3) δ 27.87,

29.43, 33.31, 55.59, 67.09, 99.52, 105.62, 121.15, 123.24, 129.99, 131.34, 145.33, 146.23,

157.62, 160.80.

4-(4-Bromobutoxy)-2-isopropoxy-4'-nitro-1,1'-biphenyl. (3.48b) Yellow powder, 0.24 g, yield

1 87% starting from 0.18 g 3.47b (0.67 mmol); H NMR (400 MHz, CDCl3) δ 1.31 (d, J = 6.1 Hz,

210

6H), 1.96-2.04 (m, 2H), 2.08-2.16 (m, 2H), 3.53 (t, J = 6.5 Hz, 2H), 4.06 (t, J = 6.1 Hz, 2H), 4.54

(sep, J = 6.1 Hz, 1H), 6.57-6.60 (m, 2H), 7.26-7.30 (m, 1H), 7.68-7.72 (m, 2H), 8.22-8.26 (m,

13 2H); C NMR (100 MHz, CDCl3) δ 21.98, 27.88, 29.44, 33.33, 67.06, 70.97, 101.98, 105.92,

122.20, 123.10, 129.99, 131.48, 145.73, 146.11, 155.96, 160.60.

4-((6-Bromohexyl)oxy)-2-methoxy-4'-nitro-1,1'-biphenyl. (3.48c) Yellowish white powder, 0.39

1 g, yield 83% starting from 0.28 g 3.47c (1.14 mmol); H NMR (400 MHz, CDCl3) δ 1.52-1.60

(m, 4H), 1.83-1.98 (m, 4H), 3.46 (t, J = 6.7 Hz, 2H), 3.85 (s, 3H), 4.05 (t, J = 6.4 Hz, 2H), 6.58-

6.62 (m, 2H), 7.26-7.29 (m, 1H), 7.67-7.70 (m, 2H), 8.23-8.27 (m, 2H); 13C NMR (100 MHz,

CDCl3) δ 25.31, 27.91, 29.07, 32.66, 33.75, 55.58, 67.98, 99.55, 105.64, 120.96, 123.24, 129.98,

131.32, 145.41, 146.20, 157.61, 161.01.

4-((6-Bromohexyl)oxy)-2-isopropoxy-4'-nitro-1,1'-biphenyl. (3.48d) Yellow powder, 0.26 g,

1 yield 88% starting from 0.18 g 3.47b (0.67 mmol); H NMR (400 MHz, CDCl3) δ 1.31 (d, J =

6.1 Hz, 6H), 1.53-1.58 (m, 4H), 1.81-1.89 (m, 2H), 1.90-1.97 (m, 2H), 3.46 (t, J = 6.7 Hz, 2H),

4.03 (t, J = 6.3 Hz, 2H), 4.54 (sep, J = 6.1 Hz, 1H), 6.57-6.61 (m, 2H), 7.26-7.29 (m, 1H), 7.68-

13 7.72 (m, 2H), 8.22-8.26 (m, 2H); C NMR (100 MHz, CDCl3) δ 21.99, 25.31, 27.91, 29.07,

32.66, 33.75, 67.94, 70.94, 102.00, 105.95, 122.01, 123.10, 129.98, 131.45, 145.80, 146.08,

155.95, 160.81.

4-((6-Bromohexyl)oxy)-2,2'-dimethoxy-4'-nitro-1,1'-biphenyl. (3.48e) Yellow powder, 0.32 g,

1 yield 91% starting from 0.22 g 3.47d (0.79 mmol); H NMR (400 MHz, CDCl3) δ 1.53-1.60 (m,

4H), 1.81-1.89 (m, 2H), 1.90-1.98 (m, 2H), 3.46 (t, J = 6.7 Hz, 2H), 3.78 (s, 3H), 3.89 (s, 3H),

4.04 (t, J = 6.3 Hz, 2H), 6.56-6.59 (m, 2H), 7.14-7.18 (m, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.81 (d,

211

13 J = 2.1 Hz, 1H), 7.89 (dd, J = 8.3, 2.1 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.35, 27.92,

29.12, 32.68, 33.78, 55.64, 56.12, 67.86, 99.37, 104.98, 105.92, 115.58, 118.04, 131.48, 132.01,

134.88, 147.86, 157.53, 157.81, 160.65.

4-((6-Bromohexyl)oxy)-2,2'-diisopropoxy-4'-nitro-1,1'-biphenyl. (3.48f) Yellow oil, 0.28 g, yield

1 90% starting from 0.21 g of 3.47d (0.63 mmol); H NMR (400 MHz, CDCl3) δ 1.21 (d, J = 6.0

Hz, 6H), 1.28 (d, J = 6.0 Hz, 6H), 1.53-1.58 (m, 4H), 1.81-1.89 (m, 2H), 1.90-1.98 (m, 2H), 3.46

(t, J = 6.7 Hz, 2H), 4.02 (t, J = 6.4 Hz, 2H), 4.42 (sep, J = 6.0 Hz, 1H), 4.54 (sep, J = 6.0 Hz,

1H), 6.52-6.56 (m, 2H), 7.16 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 2.1 Hz,

13 1H), 7.83 (dd, J = 8.3, 2.1 Hz, 1H); C NMR (100 MHz, CDCl3) δ 21.82, 22.01, 25.36, 27.95,

29.15, 32.68, 33.77, 67.81, 70.88, 71.44, 101.91, 105.11, 108.84, 115.09, 119.79, 131.84, 132.50,

136.53, 147.48, 155.84, 156.15, 160.13.

212

Synthesis of biphenoxyalkyl AIA azole hybid intermediates (3.49a-f)

Compounds 3.49a-f were synthesized from 3.48a-f (0.52-0.92 mmol) following the general synthesis of compounds 3.10a-m.

1-(4-((2-Methoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)butyl)-1H-imidazole. (3.49a) Yellow

1 powder, 0.27 g, yield 80% starting from 0.35 g 3.49a (0.92 mmol); H NMR (400 MHz, CDCl3)

δ 1.80-1.88 (m, 2H), 2.00-2.09 (m, 2H), 3.84 (s, 3H), 4.02-4.10 (m, 4H), 6.56-6.60 (m, 2H), 6.97

(s, 1H), 7.11 (s, 1H), 7.26-7.29 (m, 1H), 7.54 (s, 1H), 7.65-7.70 (m, 2H), 8.22-8.27 (m, 2H); 13C

NMR (100 MHz, CDCl3) δ 26.34, 28.05, 46.75, 55.60, 67.32, 99.47, 105.55, 118.72, 121.29,

123.24, 129.67, 130.00, 131.37, 137.11, 145.26, 146.26, 157.63, 160.65.

1-(4-((2-Isopropoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)butyl)-1H-imidazole. (3.49b) Yellow

1 powder, 0.19 g, yield 85% starting from 0.23 g 3.48b (0.56 mmol); H NMR (300 MHz, CDCl3)

213

δ 1.30 (d, J = 6.0 Hz, 6H), 1.77-1.88 (m, 2H), 1.98-2.09 (m, 2H), 3.99-4.09 (m, 4H), 4.52 (sep, J

= 6.0 Hz, 1H), 6.53-6.58 (m, 2H), 6.96 (s, 1H), 7.09 (s, 1H), 7.26-7.30 (m, 1H), 7.53 (s, 1H),

13 7.66-7.71 (m, 2H), 8.20-8.25 (m, 2H); C NMR (75 MHz, CDCl3) δ 21.96, 26.33, 28.05, 46.75,

67.26, 70.96, 101.89, 105.77, 118.75, 122.28, 123.12, 129.63, 129.99, 131.51, 137.11, 145.66,

146.10, 155.96, 160.45.

1-(6-((2-Methoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole. (3.49c) Yellowish white powder, 0.25 g, yield 74% starting from 0.35 g 3.48c (0.85 mmol); 1H NMR (400 MHz,

CDCl3) δ 1.36-1.46 (m, 2H), 1.50-1.60 (m, 2H), 1.78-1.90 (m, 4H), 3.84 (s, 3H), 3.95-4.05 (m,

4H), 6.56-6.60 (m, 2H), 6.93 (s, 1H), 7.08 (s, 1H), 7.25-7.29 (m, 1H), 7.49 (s, 1H), 7.64-7.70 (m,

13 2H), 8.22-8.26 (m, 2H); C NMR (100 MHz, CDCl3) δ 25.65, 26.34, 29.06, 31.02, 46.92, 55.58,

67.85, 99.50, 105.63, 118.75, 121.00, 123.23, 129.51, 129.98, 131.33, 137.09, 145.37, 146.21,

157.61, 160.94.

1-(6-((2-Isopropoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole. (3.49d) Yellow oil,

1 0.21 g, yield 90% starting from 0.24 g 3.48d (0.55 mmol); H NMR (400 MHz, CDCl3) δ 1.30

(d, J = 6.1 Hz, 6H), 1.36-1.44 (m, 2H), 1.50-1.59 (m, 2H), 1.78-1.89 (m, 4H), 3.94-4.03 (m, 4H),

4.53 (sep, J = 6.1 Hz, 1H), 6.55-6.59 (m, 2H), 6.93 (s, 1H), 7.08 (s, 1H), 7.26-7.29 (m, 1H), 7.49

13 (s, 1H), 7.67-7.71 (m, 2H), 8.21-8.25 (m, 2H); C NMR (100 MHz, CDCl3) δ 21.97, 25.64,

26.34, 29.07, 31.03, 46.92, 67.81, 70.95, 101.97, 105.93, 118.74, 122.05, 123.10, 129.50, 129.97,

131.46, 137.08, 145.77, 146.08, 155.95, 160.74.

1-(6-((2,2'-Dimethoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole. (3.49e) Yellow

1 oil, 0.22 g, yield 81% starting from 0.28 g 3.48e (0.63 mmol); H NMR (400 MHz, CDCl3) δ

214

1.36-1.46 (m, 2H), 1.50-1.59 (m, 2H), 1.78-1.90 (m, 4H), 3.77 (s, 3H), 3.88 (s, 3H), 3.95-4.03

(m, 4H), 6.54-6.57 (m, 2H), 6.93 (s, 1H), 7.08 (s, 1H), 7.13-7.17 (m, 1H), 7.39 (d, J = 8.3 Hz,

1H), 7.49 (s, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.88 (dd, J = 8.3, 2.0 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 25.67, 26.33, 29.10, 31.04, 46.93, 55.64, 56.11, 67.72, 99.32, 104.97, 105.92, 115.57,

118.09, 118.75, 129.48, 131.49, 132.00, 134.84, 137.07, 147.86, 157.52, 157.82, 160.59.

1-(6-((2,2'-Diisopropoxy-4'-nitro-[1,1'-biphenyl]-4-yl)oxy)hexyl)-1H-imidazole. (3.49f) Yellow

1 oil, 0.23 g, yield 91% starting from 0.26 g of 3.48f (0.52 mmol); H NMR (400 MHz, CDCl3) δ

1.21 (d, J = 6.0 Hz, 6H), 1.27 (d, J = 6.0 Hz, 6H), 1.36-1.45 (m, 2H), 1.50-1.59 (m, 2H), 1.78-

1.90 (m, 4H), 3.95-4.01 (m, 4H), 4.41 (sep, J = 6.0 Hz, 1H), 4.54 (sep, J = 6.0 Hz, 1H), 6.50-

6.56 (m, 2H), 6.93 (s, 1H), 7.08 (s, 1H), 7.14-7.18 (m, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.49 (s,

13 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.84 (dd, J = 8.3, 2.0 Hz, 1H); C NMR (100 MHz, CDCl3) δ

21.81, 22.00, 25.68, 26.36, 29.15, 31.04, 46.93, 67.68, 70.90, 71.43, 101.88, 105.10, 108.80,

115.08, 118.76, 119.85, 129.48, 131.86, 132.50, 136.47, 137.07, 147.50, 155.83, 156.16, 160.06.

215

Synthesis of biphenoxyalkyl AIA azole hybid intermediates (3.50a-f)

Compounds 3.50a-f were synthesized from 3.49a-f (0.45-0.65 mmol) following the general synthesis of compounds 3.11a-m.

4'-(4-(1H-imidazol-1-yl)butoxy)-2'-methoxy-[1,1'-biphenyl]-4-amine. (3.50a) Buff powder, 0.20

1 g, yield 91 starting from 0.24 g 3.49a (0.65 mmol); H NMR (400 MHz, CDCl3) δ 1.78-1.86 (m,

2H), 1.99-2.07 (m, 2H), 3.62 (brs, 2H), 3.80 (s, 3H), 4.02 (t, J = 6.0 Hz, 2H), 4.06 (t, J = 7.1 Hz,

2H), 6.50-6.54 (m, 2H), 6.71-6.75 (m, 2H), 6.97 (s, 1H), 7.10 (s, 1H), 7.20 (d, J = 8.0 Hz, 1H),

13 7.30-7.33 (m, 2H), 7.53 (s, 1H); C NMR (100 MHz, CDCl3) δ 26.39, 28.12, 46.77, 55.55,

67.19, 99.41, 105.04, 114.80, 118.77, 123.93, 128.51, 129.60, 130.29, 130.83, 137.13, 145.09,

157.44, 158.82.

216

4'-(4-(1H-imidazol-1-yl)butoxy)-2'-isopropoxy-[1,1'-biphenyl]-4-amine. (3.50b) Buff powder,

1 0.15 g, yield 90% starting from 0.18 g 3.49b (0.45 mmol); H NMR (400 MHz, CDCl3) δ 1.27

(d, J = 6.0 Hz, 6H), 1.78-1.85 (m, 2H), 1.99-2.07 (m, 2H), 3.53 (brs, 2H), 4.00 (t, J = 6.0 Hz,

2H), 4.06 (t, J = 7.1 Hz, 2H), 4.41 (sep, J = 6.0 Hz, 1H), 6.51-6.55 (m, 2H), 6.70-6.75 (m, 2H),

6.96 (s, 1H), 7.10 (s, 1H), 7.20-7.23 (m, 1H), 7.32-7.36 (m, 2H), 7.53 (s, 1H); 13C NMR (100

MHz, CDCl3) δ 22.05, 26.37, 28.12, 46.78, 67.16, 70.93, 102.91, 105.87, 114.68, 118.77,

125.27, 129.04, 129.55, 130.28, 130.99, 137.11, 144.78, 155.72, 158.52.

4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2'-methoxy-[1,1'-biphenyl]-4-amine. (3.50c) Buff powder,

1 0.19 g, yield 94% starting from 0.22 g 3.49c (0.55 mmol); H NMR (400 MHz, CDCl3) δ 1.35-

1.44 (m, 2H), 1.50-1.58 (m, 2H), 1.77-1.89 (m, 4H), 3.65 (brs, 2H), 3.80 (s, 3H), 3.94-4.01 (m,

4H), 6.51-6.56 (m, 2H), 6.71-6.75 (m, 2H), 6.93 (s, 1H), 7.08 (s, 1H), 7.20 (d, J = 8.1 Hz, 1H),

13 7.28-7.34 (m, 2H), 7.49 (s, 1H); C NMR (100 MHz, CDCl3) δ 25.67, 26.35, 29.15, 31.04,

46.94, 55.54, 67.70, 99.45, 105.09, 114.81, 118.76, 123.66, 128.61, 129.46, 130.29, 130.79,

137.08, 145.04, 157.40, 157.09.

4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2'-isopropoxy-[1,1'-biphenyl]-4-amine. (3.50d) Yellowish white powder, 0.16 g, yield 86% starting from 0.20 g 3.49d (0.47 mmol); 1H NMR (400 MHz,

CDCl3) δ 1.27 (d, J = 6.1 Hz, 6H), 1.36-1.45 (m, 2H), 1.50-1.59 (m, 2H), 1.77-1.89 (m, 4H),

2.74 (brs, 2H), 3.95-4.00 (m, 4H), 4.41 (sep, J = 6.1 Hz, 1H), 6.51-6.55 (m, 2H), 6.70-6.74 (m,

2H), 6.94 (s, 1H), 7.10 (s, 1H), 7.19-7.22 (m, 1H), 7.33-7.36 (m, 2H), 7.57 (s, 1H); 13C NMR

(100 MHz, CDCl3) δ 22.07, 25.65, 26.33, 29.13, 30.99, 47.12, 67.67, 70.89, 102.94, 105.95,

114.69, 118.87, 125.00, 128.89, 129.14, 130.27, 130.94, 136.90, 144.72, 155.68, 158.78.

217

4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2,2'-dimethoxy-[1,1'-biphenyl]-4-amine. (3.50e) Buff

1 powder, 0.19 g, yield 93% starting from 0.22 g 3.49e (0.51 mmol); H NMR (400 MHz, CDCl3)

δ 1.35-1.45 (m, 2H), 1.50-1.58 (m, 2H), 1.76-1.88 (m, 4H), 3.42 (brs, 2H), 3.74 (s, 3H), 3.76 (s,

3H), 3.94-4.01 (m, 4H), 6.33-6.36 (m, 2H), 6.51 (dd, J = 8.2, 2.4 Hz, 1H), 6.53 (d, J = 2.2 Hz,

1H), 6.93 (s, 1H), 7.00-7.03 (m, 1H), 7.09 (s, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.50 (s, 1H); 13C

NMR (100 MHz, CDCl3) δ 25.69, 26.35, 29.17, 31.05, 46.96, 55.57, 55.67, 67.58, 98.95, 99.34,

104.67, 107.04, 117.83, 118.79, 120.51, 129.42, 132.00, 132.26, 137.09, 146.95, 158.00, 158.17,

159.25.

4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2,2'-diisopropoxy-[1,1'-biphenyl]-4-amine. (3.50f) Waxy yellow oil, 0.18 g, yield 87% starting from 0.22 g of 3.49f (0.45 mmol); 1H NMR (400 MHz,

CDCl3) δ 1.18 (t, J = 6.2 Hz, 12H), 1.35-1.45 (m, 2H), 1.49-1.58 (m, 2H), 1.76-1.89 (m, 4H),

3.06 (brs, 2H), 3.94-3.99 (m, 4H), 4.22-4.35 (m, 2H), 6.31-6.36 (m, 2H), 6.48-6.53 (m, 2H), 6.93

(s, 1H), 7.03-7.07 (m, 1H), 7.08 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H), 7.50 (s, 1H); 13C NMR (100

MHz, CDCl3) δ 22.12, 22.15, 25.68, 26.36, 29.19, 31.04, 46.99, 67.57, 70.89, 70.99, 102.93,

102.99, 105.39, 107.49, 118.83, 120.10, 122.56, 129.32, 132.58, 132.79, 137.04, 146.20, 156.35,

156.47, 158.64.

218

Synthesis of biphenoxyalkyl AIA azole hybrids (3.51a-f)

Compounds 3.51a-f were synthesized from 3.50a-f (0.31-0.47 mmol) following the general synthesis of compounds 3.12a-n.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-2'-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide. (3.51a)

Yellow powder, 148 mg, yield 71% starting from 160 mg 3.50a (0.47 mmol); mp 139-141°C;

1 H NMR (400 MHz, CDCl3) δ 1.79-1.87 (m, 2H), 2.00-2.08 (m, 2H), 3.84 (s, 3H), 4.03 (t, J =

6.0 Hz, 2H, overlapped), 4.06 (t, J = 7.1 Hz, 2H, overlapped), 5.94 (brs, 2H), 6.53-6.57 (m, 2H),

6.97 (4.03 (t, J = 1.2 Hz, 1H), 7.05-7.11 (m, 3H), 7.26-7.29 (m, 1H), 7.41 (ddd, J = 7.5, 4.8, 1.1

Hz, 1H), 7.50-7.56 (m, 3H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.61 (ddd, J

13 = 4.8, 1.7, 0.9 Hz, 1H); C NMR (100 MHz, CDCl3) δ 26.39, 28.12, 46.76, 55.61, 67.21, 99.47,

105.18, 118.76, 121.22, 121.65, 123.67, 125.11, 129.63, 130.49, 131.15, 132.96, 136.78, 137.13,

219

147.85, 148.28, 151.67, 152.58, 157.50, 159.16; HRMS (ESI) m/z (M +H)+ calcd for

C26H28N5O2, 442.22375; found, 442.22559; Anal. Calcd for C26H27N5O2: C, 70.73; H, 6.16; N,

15.86. Found: C, 70.69; H, 6.27; N, 15.69.

N-(4'-(4-(1H-imidazol-1-yl)butoxy)-2'-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide.

(3.51b) buff powder, 95 mg, yield 53% starting from 140 mg 3.50b (0.38 mmol); mp 122-124

1 °C; H NMR (700 MHz, CDCl3) δ 1.29 (d, J = 6.1 Hz, 6H), 1.80-1.84 (m, 2H), 2.01-2.06 (m,

2H), 4.01 (d, J = 6.0 Hz, 2H), 4.06 (d, J = 7.2 Hz, 2H), 4.44 (sep, J = 6.1 Hz, 1H), 6.55-6.57 (m,

2H), 6.97 (s, 1H), 7.05-7.08 (m, 2H), 7.10 (s, 1H), 7.28 (d, J = 9.0 Hz, 1H), 7.42 (ddd, J = 7.3,

4.8, 0.6 Hz, 1H), 7.53 (s, 1H), 7.54-7.56 (m, 2H), 7.84 (td, J = 7.7, 1.6 Hz, 1H), 8.47 (d, J = 7.9

13 Hz, 1H), 8.60 (d, J = 4.8 Hz, 1H); C NMR (175 MHz, CDCl3) δ 22.09, 26.38, 28.13, 46.78,

67.19, 71.08, 102.87, 105.93, 118.77, 121.08, 121.65, 125.03, 125.17, 129.61, 130.56, 131.25,

133.51, 136.82, 137.14, 147.88, 151.54, 152.67, 155.86, 158.90; HRMS (ESI) m/z (M +H)+ calcd for C28H32N5O2, 470.25505; found, 470.25513; Anal. Calcd for C28H31N5O2: C, 71.62; H,

6.65; N, 14.91. Found: C, 71.51; H, 6.60; N, 14.76.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2'-methoxy-[1,1'-biphenyl]-4-yl)picolinimidamide.

(3.51c) Yellow powder, 143 mg, yield 70% starting from 160 mg 3.50c (0.43 mmol); mp 135-

1 137 °C; H NMR (400 MHz, CDCl3) δ 1.37-1.45 (m, 2H), 1.51-1.60 (m, 2H), 1.78-1.89 (m,

4H), 3.84 (s, 3H), 3.95-4.03 (m, 4H), 5.93 (brs, 2H), 6.54-6.58 (m, 2H), 6.93 (t, J = 1.1 Hz, 1H),

7.05-7.10 (m, 3H), 7.26-7.29 (m, 1H), 7.41 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.49 (s, 1H), 7.52-

7.56 (m, 2H), 7.84 (td, J = 7.7, 1.8 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.60 (ddd, J = 4.8, 1.6, 0.9

13 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.68, 26.36, 29.15, 31.05, 46.93, 55.59, 67.73, 99.50,

220

105.22, 118.74, 121.19, 121.64, 123.41, 125.09, 129.51, 130.49, 131.10, 133.04, 136.76, 137.08,

147.84, 147.27, 151.70, 152.55, 157.46, 159.42; HRMS (ESI) m/z (M +H)+ calcd for

C28H32N5O2, 470.25505; found, 470.25662; Anal. Calcd for C28H31N5O2: C, 71.62; H, 6.65; N,

14.91. Found: C, 71.66; H, 6.82; N, 14.78.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2'-isopropoxy-[1,1'-biphenyl]-4-yl)picolinimidamide.

(3.51d) Buff powder, 40 mg, yield 23% starting from 140 mg 3.50d (0.35 mmol); mp 98-102 °C;

1 H NMR (400 MHz, CDCl3) δ 1.29 (d, J = 6.0 Hz, 6H), 1.36-1.46 (m, 2H), 1.50-1.59 (m, 2H),

1.78-1.89 (m, 4H), 3.96-4.03 (m, 4H), 4.45 (sep, J = 6.0 Hz, 1H), 5.91 (brs, 2H), 6.54-6.59 (m,

2H), 6.93 (t, J = 1.2 Hz, 1H), 7.02-7.07 (m, 2H), 7.08 (d, J = 1.0 Hz, 1H), 7.28 (d, J = 9.0 Hz,

1H), 7.42 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 7.49 (s, 1H), 7.53-7.57 (m, 2H), 7.84 (td, J = 7.7, 1.7

Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.60 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H); 13C NMR (100 MHz,

CDCl3) δ 22.09, 25.67, 26.36, 29.14, 31.05, 46.94, 67.71, 71.06, 102.94, 106.03, 118.75, 121.00,

121.60, 124.83, 125.09, 129.47, 130.54, 131.19, 133.50, 136.77, 137.07, 147.84, 148.15, 151.70,

+ 152.51, 155.83, 159.15; HRMS (ESI) m/z (M +H) calcd for C30H36N5O2, 498.28635; found,

498.28465; Anal. Calcd for C30H35N5O2: C, 72.41; H, 7.09; N, 14.07. Found: C, 72.16; H, 7.17;

N, 13.97.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2,2'-dimethoxy-[1,1'-biphenyl]-4-yl)picolinimidamide.

(3.51e) Buff powder, 115 mg, yield 65% starting from 140 mg 3.50e (0.35 mmol); mp 131-133

1 °C; H NMR (400 MHz, CDCl3) δ 1.36-1.46 (m, 2H), 1.51-1.60 (m, 2H), 1.78-1.89 (m, 4H),

3.78 (s, 3H), 3.79 (s, 3H), 3.95-4.02 (m, 4H), 5.90 (brs, 2H), 6.54 (dd, J = 8.2, 2.3 Hz, 1H) 6.56

(d, J = 2.3 Hz, 1H), 6.66-6.71 (m, 2H), 6.93 (s, 1H), 7.08 (s, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.22-

221

7.25 (m, 1H), 7.42 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.50 (s, 1H), 7.84 (d, J = 7.8, 1.7 Hz, 1H),

13 8.47 (d, J = 7.9 Hz, 1H), 8.61 (d, J = 4.4 Hz, 1H); C NMR (100 MHz, CDCl3) δ 25.70, 26.35,

29.17, 31.06, 46.95, 55.70, 55.74, 67.60, 99.44, 104.80, 105.11, 112.97, 118.75, 120.39, 121.64,

122.25, 125.16, 129.47, 132.01, 132.41, 136.81, 137.08, 147.88, 150.00, 151.58, 152.67, 158.12,

+ 158.17, 159.48; HRMS (ESI) m/z (M +H) calcd for C29H34N5O3, 500.26562; found, 500.26785;

Anal. Calcd for C29H33N5O3: C, 69.72; H, 6.66; N, 14.02. Found: C, 69.49; H, 6.80; N, 13.75.

N-(4'-((6-(1H-imidazol-1-yl)hexyl)oxy)-2,2'-diisopropoxy-[1,1'-biphenyl]-4- yl)picolinimidamide. (3.51f) yellow powder, 92 mg, yield 53% starting from 140 mg 3.50f (0.31

1 mmol); mp 129-132 °C; H NMR (700 MHz, CDCl3) δ 1.20 (d, J = 6.1 Hz, 12H), 1.38-1.43 (m,

2H), 1.52-1.57 (m, 2H), 1.79-1.87 (m, 4H), 3.95-4.00 (m, 4H), 4.33 (sep, J = 6.1 Hz, 1H), 4.37

(sep, J = 6.1 Hz, 1H), 6.52-6.56 (m, 2H), 6.63-6.68 (m, 2H), 6.93 (s, 1H), 7.08 (s, 1H), 7.22 (d, J

= 8.0 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.40-7.43 (m, 1H), 7.49 (s, 1H), 7.83-7.86 (m, 1H), 8.46

13 (d, J = 7.8 Hz, 1H), 8.60 (d, J = 4.7 Hz, 1H) ; C NMR (100 MHz, CDCl3) δ 22.15, 22.17,

25.71, 26.39, 29.21, 31.07, 46.95, 67.59, 70.75, 71.19, 103.01, 105.46, 108.55, 113.13, 118.77,

121.56, 122.48, 124.20, 125.13, 129.50, 132.46, 133.03, 136.80, 137.09, 147.89, 149.63, 151.66,

+ 152.65, 156.51, 158.88; HRMS (ESI) m/z (M +H) calcd for C33H42N5O3, 556.32822; found,

556.32831; Anal. Calcd for C33H41N5O3: C, 71.32; H, 7.44; N, 12.60. Found: C, 71.02; H, 7.51;

N, 12.36.

222

4.3. Biological Assays

Axenic amastigote assay. Axenic amastigote assays were carried out essentially as described previously with some modifications [191]. Briefly, L. donovani Ld1s strain axenic amastigotes were maintained in the medium described earlier [192]. Parasites were plated at a cell density of

106 parasites/mL in a volume of 60 µL/well together with twofold serial dilutions of cyanine compounds or the control drug pentamidine and allowed to incubate for approximately 72 hours at 37 °C in a 5% CO2 atmosphere. Following this incubation, 12 µL of CellTiter reagent was added and the assay was allowed to continue for an additional 5-6 hours. In order to kill parasites (which was required due to the need to quantitate the assay in a BSL1 lab), 8 µL of

10% SDS in water was added and incubation was continued for 1 hour before reading the absorbance at 490 nm with the aid of a SpectraMax microplate reader (Molecular Devices,

Sunnyvale, CA) to aid in determining IC50 values.

J774 macrophage toxicity assay. The assay to determine the toxicity of cyanines and hybrid compounds to J774 murine macrophages was performed as described by Zhu et al. [193], with the modification that absolute CC50 values were calculated as described for IC50 values by Joice et al. [160].

HepG2 cytotoxicity assay. The HepG2 assay was conducted as described previously [194] with minor adaptations. HepG2 cells were maintained in a complete MEM Alpha medium (MEM

223

Alpha [Life Technologies] with 10% heat-inactivated FBS [Sigma-Aldrich], 50 U/mL penicillin, and 50 ug/mL streptomycin [Life Technologies]). Cells were trypsinized with TrypLE Express

(Life Technologies) and plated in 96 well plates at a density of 5 × 103 cells/well in 100 µL volume and allowed to adhere overnight at 37 °C in a 5% CO2 atmosphere. The following day the medium was replaced with medium containing either compound or control drug and allowed to incubate for approximately 72 hours at 37 °C in a 5% CO2 atmosphere. Following this incubation, medium was removed and replaced with 100 µL/well of fresh medium and 25

µL/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL in sterile PBS), the the plates were incubated for an additional 2-3 hours. After incubation, 100 µL of SDS lysis buffer (100 mg/mL SDS in 50% aqueous DMF) was added to lyse the cells. After an additional 3-5 hour incubation the absorbance was read at 570 nm with the aid of a

SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA). IC50 determinations were made as previously described [160].

L. donovani-infected macrophage assay. An intracellular L. donovani high-content imaging assay was carried out as described previously [160]. Briefly, peritoneal macrophages from CD-1 mice were plated in 96-well plates (Corning, Corning, NY) at a density of 105 macrophages/well in macrophage medium (RPMI 1640 with GlutaMAX [Life Technologies, Grand Island, NY],

10% heat-inactivated FBS [Sigma-Aldrich], 100 U/ml penicillin, and 100 µg/mL streptomycin

[Life Technologies], pH 7.4). The macrophages were allowed to adhere overnight at 37 °C in a

5% CO2 atmosphere and infected the following day with stationary-phase L. donovani LV82

224 promastigotes at a parasite/macrophage ratio of 5:1. The infection proceeded overnight at 37 °C in a 5% CO2 atmosphere and extracellular parasites were washed away with HBSS (Life

Technologies) the following morning. Fresh medium containing either compound or the standard drug amphotericin B was added to the plate and incubation was continued for approximately 72 hours at 37 °C in a 5% CO2 atmosphere. Following incubation with compound, the plates were washed with PBS, fixed with formalin, and stained with DAPI as described in detail earlier [160]. The plates were then imaged and analyzed using a Thermo

Scientific ArrayScan XTI live high-content platform (Thermo Fisher Scientific, Pittsburgh, PA) and IC50 values were calculated as described previously [160].

Evaluation of 3.24c in a murine visceral leishmaniasis model. The in vivo antileishmanial efficacy of this hybrid compound was assessed in L. donovani-infected female mice essentially as described earlier [191]. In the present study, the uninfected vehicle control group received

PEG-400, miltefosine was dissolved in water, and compound 3.24c was dissolved in 50%

PEG400/50% water. Administration was by oral gavage in each case.

UV-vis thermal melting (Tm). DNA thermal melting experiments were performed on a Cary 300

Bio UV-vis spectrophotometer (Varian). The concentration of each hairpin DNA sequence was 3

μM in buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.4) using 1 cm quartz cuvettes. The solutions of DNA and ligands were tested with the ratio of 2:1 [ligand]:[DNA]. All samples were

225 increased to 95 °C and cooled down to 25 °C slowly before each experiment. The spectrophotometer was set at 260 nm with a 0.5 °C/min increase beginning at 25 °C, which is below the DNA melting temperature and ending above it at 95 °C. The absorbance of the buffer was subtracted, and a graph of normalized absorbance versus temperature was created using

KaleidaGraph 4.0 software. The ΔTm values were calculated using a combination of the derivative function and estimation from the normalized graphs.

Circular dichroism (CD). Circular dichroism experiments were performed on a Jasco J-810 CD spectrometer in a 1 cm quartz cuvette at 25 °C. A buffer scan as a baseline was collected first in the same cuvette and subtracted from the scan of following samples. The DNA (10 µM), in buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.4) was added to the cuvette prior to the titration experiments and then the compound was added to the DNA solution and incubated for 5 min to achieve equilibrium binding for the DNA-ligand complex formation. For each titration point, four spectra were averaged from 500 to 230 nm wavelength with scan speed 50 nm/min, with a response time of 1 s. Baseline-subtracted graphs were created using KaleidaGraph 4.0 software.

226

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Appendix

NMR spectra of cyanines and arylimidamide azole hybrids

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Ethyl acetate peaks = 14.21, 21.06, 60.40 Diethylether peaks = 15.29, 65.86

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