Chapter 1: Introduction

1.1 Open Source Science

Open source science is a collaborative method in which the scientific community shares information about research projects online. Anyone with access to the internet is able to contribute to the project via a web-based format. (Hooker 2007, Todd 2007)

The idea of open source development isn’t novel. Open source development was originally used in computer software development, where users were able to contribute small pieces toward a bigger picture, such as the Linux project (Linux 2007).

NASA’s Clickworkers project is an example of a successful open source science project where volunteers helped to identify craters on Mars (NASA 2001). The website provides some minimal training for users to help them identify craters. In 2001 NASA was able to produce an age map of Mars by identifying the craters as new, aged, or ghost based on the input of all of the volunteers. NASA is currently beginning a similar program to identify craters on asteroids.

Open source science recently has moved toward the investigation of drugs that could cure diseases that are endemic to third world nations (Taylor 2006). Open source science is well suited for research in these areas because many pharmaceutical companies will not invest in these drugs because of poor profit return (Everts 2006). Unfortunately, many people afflicted with these diseases cannot afford the medications to treat their condition. The motivation for using open source science to research drugs for tropical diseases such as malaria and tuberculosis is to eventually produce a library of drugs that are made readily available for development.

Researchers who participate in open source science give up their patent rights because the information is made public as soon as it is posted. However, the goal of open source science is to contribute to the greater good of human society, worth more than any patent. It has often been argued that open source science could provide a pipeline for non-profit pharmaceutical companies such as One World Health (One World Health 2007). One World Health relies on donations of new drug leads from research communities in academia and industry. One World Health was recently approved by the Indian government for its new drug paromomycin, an antibiotic that cures visceral leishmaniasis (VL).

1.2 Open Notebook Science
In our group, Open Notebook Science is a means of communicating with the scientific community in a manner as transparent as possible about the research that is being conducted in our lab (Bradley 2006). At (Bradley 2007A), information about current projects is posted for everyone to read in a blog.

Figure 1.1 Useful Chemistry Blog

Visitors can offer feedback about posts concerning our experiments. From this page, they can also be redirected to our molecules blog (Bradley 2007B), where all molecules used and produced in our experiments are posted (Figure 1.2). Some detail such as NMR data, SMILES code, and commercial availability are provided with each post. Links to our experiments are also provided with molecules used in any experiments.

Figure 1.2 Useful Chemistry Molecules Blog.

All experiments are posted at (Figure 1.3). The wiki acts as an online laboratory notebook. All raw experimental data including a log are updated concurrently with the experiment. The post is arranged just as it would be in a paper notebook, with sections including objective, procedure, and discussion. In essence, a visitor to our website could monitor an experiment and comment on it as the experiment happens.

Figure 1.3 Useful Chem Wiki

The posting of raw data in Open Notebook Science includes failed experiments, often omitted in scientific journals. This narrows the gap in interpretation of results because users gain a better understanding of the research through failed experiments. Researchers also don’t repeat failed experiments, thereby reducing the amount of time spent on a particular project.

Using Sitemeter (Sitemeter 2007) on the wiki and blogs, we could also monitor the number of users that visit our websites over a period of time. As of May 2007 we've had over 30, 000 vistors to the site and we've experienced a four fold increase in the number of monthly visitors in the past year (May 2006-May 2007) (Figures 1.4, 1.5). We could also look at the search parameters visitors are using and from where they are searching (Figure 1.6) and find that people all over the world are visiting our website.

Figure 1.4 Sitemeter can be used to monitor the total number of visitors to the site.

Figure 1.5 Graph showing the increase in monthly visitors.

Figure 1.6 Sitemeter showing the locations of the visitors to the Useful Chem Wiki.

1.3 Malaria
Malaria is an infectious disease that infects between 300-500 million people each year (Centers for Disease Control and Prevention (CDC) 2007). Up to three million people die from malaria each year, most of them children and pregnant women (CDC 2007). Forty one percent of the world’s population lives in areas where malaria is transmitted (Sachs 2002), mostly in tropical and subtropical areas. Malaria is one of the most common infectious diseases and has an enormous impact on economic development (Sachs 2002).

Many countries that suffer from epidemic outbreaks of malaria are also the some of the world’s poorest nations. Many studies have shown that malaria is not only the result of poverty, but the cause of poverty (Sachs 2002). A study in the comparison of incomes in non-malarious and malarious countries demonstrates that the average GDP was $1,526 USD in malarious countries as opposed to $8,268 USD in non-malarious countries (Tren 1999). This represents a five-fold difference. The average growth in per capita GDP in malarious nations was 0.4% per year compared to 2.3% per year in non-malarious nations (Tren 1999).

Many wealthy nations were able to eliminate the disease through intensive intervention programs such as provisions for screen doors and windows (Tren 1999). The draining of swamps also eliminated the breeding grounds for mosquitoes (Tren 1999). The spraying of DDT as an anti-malarial insecticide has helped decrease the cases of malaria in some regions; however with new environmental groups protesting the use of DDT because of its environmental effects, many countries have placed a ban on the use of DDT and other persistent organic pollutants (Tren 1999). This has led to an increase in the amount of malaria cases in many developing countries that cannot afford alternatives to DDT. It is estimated that the banning of DDT as an anti-malarial weapon will cost the economies of developing nations approximately $480 million USD (Sachs 2002).

Malaria has also affected the demographics of endemic nations (Sachs 2002). Malaria accounts for 25% of all mortality in children in Africa up to four years old (Snow 1999). School-aged children also suffer from complications of the disease because of the neurological damage caused by the disease (Sachs 2002). In one study, it was found that 13-50% of illness-related absences from school are due to the disease (Sachs 2002). This in turn feeds into the cycle of poverty with losses in job opportunities and experience.

Due to the economic losses many countries suffer, many citizens of these nations cannot afford the medication, even at a mere thirteen cents (USD) for a seven day supply of chloroquine (CDC, 2007). This in turn deters pharmaceutical companies from investing in research for the vaccine due to the lack in profitable gains. For those nations who can afford it, many of those afflicted with the disease live in rural areas where there is no access to suitable health facilities (CDC 2007).

Many of the prophylactic drugs that are currently available for treatment of malaria are unsuitable for children, leaving them especially vulnerable (CDC 2007). The use of an insecticide treated bed nets serve as the only line of defense for children under five years old (CDC 2007). In addition, the parasite has begun building a resistance to many of the current drugs. The P. falciparum and P. vivax strains of malaria have both been confirmed to be resistant to chloroquine and there are other strains that are resistant to many drugs (CDC 2007). The urgency for drugs that fight P. falciparum is pressing as it is responsible for 80-90 percent of deaths associated with malaria (CDC 2007). Despite the dismal outlook on malaria, there are many people working to solve this worldwide dilemma.

One of the organizations that are working to solve this problem is Find-A-Drug (Find-a-drug 2007). Find-A-Drug runs a non-profit computing project that serves to address problems regarding worldwide health. Find-A-Drug seeks to collaborate with experts by providing libraries of synthetic targets predicted to inhibit disease related enzymes. Researchers can then make attempts at synthesizing these molecules, as was done in this thesis work.

1.4 Diketopiperazines and Malaria
The malaria parasite synthesizes Enoyl-acyl carrier protein reductase (or ENR), an enzyme which is critical in its synthesis of fatty acids (Kuo 2003). The syntheses of these fatty acids are important for the parasite’s survival, as it is used to synthesize the cell wall.

A class of compounds called diketopiperazines has been of recent special interest because of its wide range of biological activity, including quorum sensing (Zavilgelsky 2001). These compounds may have great potential for inhibiting ENR in malaria based on docking studies made by Find-A-Drug (Bradley 2005).

1.5 Synthesis of Diketopiperazines and the Ugi Reaction
Diketopiperazines can be synthesized via a Ugi/de-BOC/Cyclize (UDC) method (Hulme 1998).


Scheme 1.1 General Reaction Scheme for the UDC Synthesis of Diketopiperazines

The first part of the synthetic pathway involves a Ugi synthesis where an aldehyde (1), an isocyanide (2), an amine (3), and a carboxylic acid (4) are added together in equimolar concentrations in methanol to form the Ugi Adduct (5). The adduct is then cyclized via a transamidation to form the diketopiperazine (6).

The Ugi reaction was first proposed by Ivar Karl Ugi in 1959. One of the benefits of the Ugi reaction is that it provides variability in the components and products because the reaction provided structurally different skeletons of the products (Ugi 2003). It was also determined that the structures of the skeletons were mainly determined based on the structures of the amines and acids (Ugi 2003).

High concentrations (0.5M-2M) of these components give the highest yields (Mironov 2003). The use of polar aprotic solvents such as DMF are advantageous (Ugi 2000), however the synthesis has been shown to work in methanol (Hulme 1998) and is even accelerated (up to 50 times greater than organic solvents) in water (Pirrung 2003).

The reaction is believed to proceed with the initial formation of the imine (7) from the aldehyde (1) and the amine (3) (Ugi 2000). This occurs when the carbon of (1) is attacked by the lone pair electrons on the nitrogen of (3).The imine is then protonated by a carboxylic acid (8) to form the iminium ion (9) and the negatively charged carboxylate ion (10). This is a necessary step in the formation of the Ugi product because it increases the electrophilicity of the C=N bond (Ugi 2000).The carboxylate anion (10) and the iminium ion (9) are then added across the carbon of the isocyanide (2). The alpha-adduct (11) is a heteroanalogue of an acid hydride, a strong acylating agent (Ugi 2000).The resulting isoamide rearranges via an intramolecular acyl transfer to give the Ugi product (12) (Ugi 2000). This acyl transfer is known as the Mumm rearrangement (Mumm 1910).

Scheme 1.2 Mechanism of the Ugi Reaction

One of the benefits of the Ugi reaction is that it is easily performed by adding all of the components together in a one-pot fashion at room temperature.

The next step in the UDC synthesis of diketopiperazines is the deprotection of the BOC group. (Scheme 1.3) Several methods have been published, many of them including using a 10% solution of trifluoracetic acid in dichloroethane (Hulme 1998). Cleavage of the BOC group of the Ugi product (5) leaves the free amine intermediate (13), which then cyclizes to a 2,5-diketopiperazine (6) (via transamidation (Hulme 1998). This is the synthetic strategy used in this work.

Scheme 1.3 Scheme for the deprotection and cyclization of the Ugi Product.

1.6 Organization of this Thesis
Chapter 2 deals with the synthesis of DOPAL (3,4-dihydroxyphenyal acetaldehyde), one of the first components of the Ugi reaction studied. Chapter 3 deals with initial Ugi synthesis attempts including the use of phenylacetaldehyde and benzylisocyanide. The Ugi reaction was then investigated in a stepwise fashion. Chapter 4 deals with the formation of the imine between the aldehyde and amine. Chapter 5 deals with the addition of the boc protected amino acid. Chapter 6 deals with the addition of isocyanide and consequently the formation and characterization of the Ugi Adduct. Chapter 7 deals with attempted cyclization of the Ugi product into diketopiperazines. Chapter 8 deals with conclusions and recommendations for future work.

1.7 Reference List

Bradley, JC 2005

Bradley, JC 2006

Bradley, JC 2007A

Bradley JC 2007B

Bradley JC 2007C

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Domling, A. Convergent and fast route to piperazines via IMCR. Org Chem. Highlights 2005 July 5

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Hooker, B. 2007

Hulme, C; Morrisette, M; Volz, F; Burns, C. The Solution Phase Synthesis of Diketopiperazine Libraries via the Ugi Reaction: Novel Application of Armstrong’s Convertible Isonitrile. Tet Lett, , 39, 1113-1116, 1998

Kuo, M., Morbidoni, H., et al. Targeting tuberculosis and malaria through inhibition of enoyl reductase. J. Biol. Chem. 278(23), 20851, 2003

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Mumm, O. Ber. Dtsch. Chem. Ges. 43, 887, 1910

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Pirrung, M., Sarma, K. Multicomponent Reactions are Accelerated in Water. J. American Chemical Society 126(2), 444-445, 2003
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Sachs, J. and Malaney, P. The economic and social burden of malaria. Nature Vol. 415, 680, 2002
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Snow, R. Craig, M., et al. Estimating mortality, morbidity and disability due to malaria among Africa’s non-pregnant population. Bull. World Health Org 77, 624-640, 1999

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Todd, M. H. Open Access and Open Source in Chemistry, Chemistry Central Journal, Vol. 1, 3, 2007. doi:10.1186/1752-153X-1-3

Tren, R. et al The Economic Costs of Malaria in South Africa. 1999

Ugi, I. and Domling, A. Multicomponent Reactions with isocyanides. Angew Chem Int. Ed., 39, 3168, 2000 DOI: 10.1002/1521-3773(20000915)39:18

Ugi, I., Werner B., Domling, A. The Chemistry of Isocyanides, their MultiComponent Reactions and their Libraries. Molecules, 8, 53-66, 2003 ISSN 1420-3049

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Zavilgelsky, G.B., and Manukhov, I.V. Quorum sensing, or how bacteria ‘talk’ to each other. Mol Bio Vol. 35 (2), 224-232, 2001