The objective of this project was to synthesize a protected spermidine derivative, which is a polyamine derivative. The spermidine that was to be synthesised is a N8 protectedspermidine derivative. Other students have studied the synthesis ofN1 protected spermidine. A retrosynthetic analysis of the required N8 protected spermidine derivative of wascarried out to devise a route to the product.
Unfortunately, this route proved unsatisfactory as due to solubility problems and had to be abandoned. However, there are other possible routes possible to synthesize the N8protected spermidine derivative. The first route by Zhang et al, provides stage 1 product3-[(3-cyanopropyl)amino] propylamine. This route is a simplenucleophilic addition reaction. The second route by Humora et al, provides the stage 2 product called NN-di-tert-butoxycarbonyl-N-(3-Cyanopropyl)-1,3-diaminopropane.
The stage 3 synthesis leading to the final product of the N8 protected spermidine was not carried out because the product is highly soluble in water and would be lost.
Since their discovery in 1678 by Antoni van Leeuwenhoek, polyamines have been the subject of extensive research which has revealed some of their biological and pathological metabolism. Recent developments include the use of transgenic mice which have tissue specific changes in polyamine concentrations. 
The naturally occurring polyamines; such as putrescine, spermine, and spermidine, are aliphatic multivalent cations which have a primary amine functional group that is completely protonated under physiological conditions. They are important building blocks in all mammalian cells.  They are derived from ornithine
by a decarboxylation/condensation process.
Polyamines are essential for cell growth, differentiation, function, and maintenance. At least one of these polyamines described above are available in every living cell. Prokaryotic and eukaryotic cells all synthesise putrescine and spermidine. Spermine synthesis is on the other hand mostly restricted to nucleated eukaryotic cells. There are essential interspecies distinctions in polyamine metabolism, in particular between eukaryotic cells, plants, and a number of bacteria and protozoa. In a few prokaryotes the polyamine synthesis is restricted to putrescine and spermidine. Whereas other prokaryotes, for example certain thermophilic bacteria, polyamines are synthesized that have even longer chains longer then spermine. Furthermore, some parasitic organisms have enzymes which are lacking in the host cells. These enzymes are used as design targets for antiparasitic compounds. Overall, prokaryotes contain greater concentration of putrescine than spermidine and no spermine. Eukaryotes normally contain minimal putrescine, but high concentrations of spermidine and spermine. 
1.2 Polyamine Structure
The terms ‘aliphatic polyamine’ or polyamine are generally used to refer the following three compounds:
[1,4-butane diamine or tetramethylenediamine],
or aminopropyl- tetramethylenediamine] 3. Spermine [N,N`-bis(3-aminopropyl)-1,4-butane diamine or
These polyamines are derived from ornithine following an initial
step.It was not until 1994 that the polyamine agmatine, derived by the decarboxylation of L-arginine, was isolated from mammalian brains and identified. (fig.1) 
Putrescine à +H3N-(CH2)4-NH3 +
Spermidine à +H3N-(CH2)3-NH-(CH2) 4-NH3+
Spermine à +H3N-(CH2)3-NH-(CH2) 4-NH-(CH2)3-NH3+
Agmatine à +H3N-(CH2)4-NH-CNH-NH 3+
Figure 1: Structure of polyamines.
1.3 Polyamine Functions
The most noticeable feature of polyamines is their polybasic environment, which provides them with more increased attraction for acidic components than that shown by Na+, K+,Mg2+ or Ca2+ . Spermidine and spermine are derived from the diamine putrescine. Spermine has the most distinctive polybasic character because its tetramine structure allows it four positive groups. The polybasicity of polyaminesenables them to bind strongly to nucleic acids and stabilise DNA.This stabilisation is achieved by the polyamine neutralizing thenegative charges on the phosphate groups of the nucleic acids, and thereby decreasing the repulsion between the DNA strands. Similarlythe interaction of polyamines with RNA influences the secondary structure of mRNA, tRNA and rRNA and therefore protein synthesisitself. Polyamines also bind to ribosomes and facilitate the association of ribosomal subunits. The significance of this isdemonstrated in eukaryotic in vitro translation systems where polyamines have been observed to increase the accuracy oftranslation. Links of polyamines with receptor proteins have also been conformed. An example of polyamine interaction with receptorproteins is that spermidine directly plugs the K+ ion channel pore as a control mechanism.
Fast growing cells have higher concentrations of polyamines. In contrast the inhibition of the polyamine biosynthetic enzymesresults in the termination of cell growth or even cell death. Abnormal polyamine metabolism is implicated in the development of tumours. This has been utilised by targeting polyamine pathway for the successful treatment of parasitic diseases. The inhibitors D,L-α-Difluoromethylornithine and MDL 73811 have been found to be for eradicating the parasites Plasmodium falciparium, Trypanosomabrucie rhodesiense, and Leishmania donovani (Table 1).
1.4 Polyamine Biosynthesis
There are exogenous and endogenous sources of polyamines for the body. They can be obtained from foods that contain relatively high concentrations of polyamines or by action of bacteria in the gut which will synthesise polyamines from dietary amino acids. This method is termed ‘exogenous’ meaning manufactured outside. The ‘endogenous’ method is by manufacture of polyamines in the liver and other
In mammalian cells, polyamines are derivatives of arginine and methionine. Putrescine is synthesised from the L-ornithine by action of ODC. Use of a particular inhibitor of ODC (DFMO) in a group of cell lines causes remarkable decrease of putrescine and spermidine, demonstrating that mammalian ODC is the main source of putrescine. Mammalian ODC is a homodimer which has two active sites formed at the interface between the two dimers. Like many decarboxylases; ODC needs pyridoxal-5-phosphate (PLP) as cofactor. The ornithine substrate could be derived from the plasma or intracellular arginine by way of action of arginase.
Putrescine is converted to spermidine and spermine via the successive action of two aminopropyl transferases, i.e. spermidine synthase and spermine synthase respectively. These two enzymes both use decarboxylated S-adenosylmethionine (dcSAM) as an aminopropyl donor but are specific with respect to their acceptors which are putrescine and spermidine respectively. SAMDC is responsible of decarboxylation of S-adenosylmethionine (SAM). dcSAM is normally very low in mammalian cells and the activities of the aminopropyltransferases are controlled by the availability of this nucleotide substrate. As a result, the making of dcSAM by means of SAMDC is an important step in polyamine production. 5′-methylthioadenosine (MTA) is the second product made at the time of the transfer of the aminopropyl group from dcSAM to putrescine or spermidine (Fig. 3). This nucleoside is then quickly degraded by MTA phosphorylase to adenine and 5′-methylthioribose-1-phosphate. The adenine is then changed into 5′-AMP by means of adenine phosphori-bosyltransferase, and 5′-methylthioribose-1-phophate is changed back to methionine. Therefore, the aminopropyl groups for spermidine or spermine synthesis originate from methionine.
Polyamines are also converted back to putrescine by polyamine oxidase and spermidine/spermine N1-acetyltransferase. Spermidine/spermine N1-acetyltransferase uses acetyl CoA to change back spermidine and spermine into N1-acetylspermidine and N1-acetylspermine respectively. The acetyl derivatives are then cleaved by polyamine
oxidase into putrescine or spermidine according to the substrate (Fig. 3). The degree of degradation is not yet known. Though, the procedure is brought on by toxic agents, by fasting, or by exposure to spermidine. The degradation may be a function of regulatory control dependent on spermidine and spermine concentrations.
1.5 Cell Growth Effects
Polyamines are important to cellular proliferation and differentiation. Children have higher levels of polyamines. Body builders think increased polyamines intake facilitates muscle growth. It was thought that the main polyamines required for growth were synthesised in the gut. Research has since proven that polyamines accumulated in the small bowel are mainly received from the food.
It not yet known how precisely polyamines stimulate growth. What is known is that they have an intense stabilising effect on DNA. One mechanism for their participation in growth processes could be through their manipulation on the activity of growth promoting genes. As polyamines derivatives are cations, they benefit from their size and electrical charge which allows them to interact with large molecules, i.e. DNA and RNA, and pass readily through e.g. phospholipid membranes.
There appears to be a close connection between polyamines, RNA and the hormone insulin. Insulin effects cell growth by stimulating ribosomes to synthesize protein. Ribosomes respond to messenger RNA. RNA copies the protein specific blueprint from DNA. Polyamines appear to stabilise and amplify the ‘message’ to produce the specific protein that is enclosed in messenger RNA.
There are two different mechanisms within the cell to stimulate growth by polyamines (Fig. 4). The first mechanism (1) entails direct influence on particular growth promoting genes. The second mechanism (2) entails the enhancement of the production of proteins required for growth. In this mechanism polyamines magnify the effects of DNA (2) and insulin (3) by stabilising messenger RNA (4). This results in a cumulative increase in protein production (5).
1.6 polyamines and Cancer
Current research has confirmed that there are raised quantities of one or more of the polyamines in the serum or urine of patients with the following conditions: leukaemia, melanoma, adenocarcinoma or lymphoma. This then led to polyamines being proposed as biochemical markers of neoplasia. Increased concentrations of polyamines in physiological fluids being diagnostic of malignant disease.  Unfortunately, increased amounts of polyamines were not limited solely to malignant conditions. Increased concentrations were also detected in body fluids of patients with; cystic fibrosis, psoriasis, or during pregnancy.
In 1958 Kosaki et al reported the presence of polyaminated compounds in cancer tissue. In those early years, polyaminated molecules and substances were recognized in all forms of life – bacteria, fungus, plants and all sorts of eukaryotic cells. They were confirmed to be essential for all types of cellular proliferation. From 1960 to 1985 there was rapid growth in studies, studies on polyamines. There were many study publications on polyaminated small molecules and compounds. This included 11 multi-chapter research review books. Tabor alone produced six broad review publications. It was expected then that knowledge and/or control of intracellular polyamine systems would have unlimited industrial and medical/clinical applications. However almost 50 years later, these expectations have not been fully realised. 
1.6.1 Apoptotic Cell Death
Eukaryotic cells have a genetic system undergo cellular suicide. This process is termed apoptosis or programmed cell death. The function of apoptosis is the complete elimination of some cells or tissues during development. The causes selection of fit clones at the time of negative selection of T-lymphocytes. The genetically damaged cells are eliminated to prevent the development of hyperplasia.Apoptosis doesn’t cause an inflammatory response, whereas necrosis does. The molecular pathways of apoptosis have been well researched.
1.6.2 Cytotoxic Effects of Polyamines
Under some conditions polyamines can have toxic effects on cells. This has occurred in cells in culture medium exposed to high levels of polyamines, or when in vivo cells have increased cellular concentrations of polyamines. The in vitro cytotoxicity of spermidine and spermine has been known since the 1970’s. It was shown that cytotoxicity of cultured cells only happened when there was ruminant serum was available in the culture medium. It did not occur if serum of other sources was used. The cytotoxicity effects of polyamines on the cultured medium containing ruminant serum could be prevented by aminoguanidine which inhibits copper amine oxidases. More research has demonstrated that cytotoxicity was caused by amine oxidase. Polyamines are relatively highly concentrated in ruminant serum. This causes spermidine and spermine to be reduced to amino aldehydes, ammonia, and hydrogen peroxide. These products have the potential to damage cells.
Intracellular accumulation of polyamines causes polyamines catabolism. This can produce cytotoxic metabolites. These metabolites can cause cell death because of shortage of detoxifying regulators. Therefore polyamine oxidation, catalysed by polyamine oxidase (PAO) (fig. 6), resulting in the production of hydrogen peroxide has implicated for causing apoptotic cell death. It was proposed from research during the 1990’s that polyamines themselves and not their oxidative metabolites can be toxic to cells.
1.6.3 Polyamine Inhibitors.
ODC has been confirmed to be the most important rate limiting and rate controlling enzymatic step in mammalian cells. In the 1970’s, difluoromethylornithine (DFMO) was found to be a powerful mechanism based inhibitor of ODC (suicide – not reversible).  DFMO has been used many times in the last 30-40 years for research:
- to elucidate the polyamine synthesis and pathways of catabolism.
- the assessment of the polyamine controlled mechanisms linking cell proliferation and differentiation.
- the inhibition of cancer cell growth.
- the improvement of human cancer therapies and also in cancer prevention programs.
- the development of new cancer chemotherapies
- even for cancer prevention
Mixtures of these four drugs reduced polyamine levels by 60 – 90% in many different cells, and slowed the proliferation of target cells, but hardly ever inhibited cell growth 100%. Most mammalian cells, including cancer cells, have membrane localised polyamine uptake and transport systems. [As soon as polyamines reduce inside a cell, these systems in polyamines from adjacent cells and the blood vascular system] The systems then take up natural polyamines and a lot of synthetic polyamine derivatives. Therefore uptake/transport systems are being used in two ways in cancer therapy:
1) to inhibit the systems and stop uptake of natural polyamines.
2) to permit the systems to be enlarged by intracellular polyamine reduction, and then provide effective polyamine derivatives which can be taken up in large amount into the cancer cell, which in turn can block cell proliferation. 
Denial of ODC enzyme activity of by DMFO makes a time dependent decrease of the cellular putrescine level, followed by decrease of spermidine. However spermine concentrations are normally not significantly decreased, and may even rise further. The reduction in putrescine concentration apparently causes the impairment of its producing enzyme, ODC.
The reduction of spermidine has three main reasons:
1) Reduced formation caused by partial availability of putrescine as substrate of spermidine synthase.
2) Thinning of polyamine pool caused by cell division and reduction in the quantity for each cell.
3) Enhanced formation of spermine caused by elevation of s-adenosylmethionine decarboxylase (AdoMetDC) activity.
The unavailability of putrescine as substrate induces AdoMetDC in DMFO treated cells and consequently hence the spermidine depletion. This eventually causes to extreme availability of dcAdoMet and leads to accumulation of spermine in the
However single enzyme inhibitors have been found to not work well. They didn’t deplete all the three polyamines to a satisfactory level and or are very toxic in humans. The results of these single enzyme inhibitors did give confirmation of the fact that inhibition of the polyamine could be a good way for the production of anti-proliferative drugs. This spurred the development and synthesis of different polyamine analogues that were made to participate or inhibit several reactions in polyamine metabolism. 
1.6.4 Polyamine Analogues
Polyamines analogues can not be fully effective in carry out the functions of natural polyamines. Therefore breakdown of the cellular functions occurs culminating in apoptosis. Cancer cells have increased levels of natural polyamines, and also have higher metabolic requirements, and/or non-regulating mechanisms. Therefore they are thought to be more susceptible to the action of polyamine analogues than normal cells. In the past, polyamine analogues were synthesised by:
- changing the chain-length of methylene groups that bridge the primary and secondary amino groups.
- adding substituents at different positions.
- alkylating the amino end groups.
Bis(ethyl) derivatives of putrescine or spermidine and spermine were synthesised by porter et al . Bis(ethyl)spermine analogues were much more efficient anti-proliferative agents than putrescine or spermidine derivatives. Polyamine analogues keep to the main pathway in the mechanism of action of polyamines.
The structures for symmetrical, terminally alkylated polyamine analogues are given in
Before 1993, no effective synthesis leading to unsymmetrically substituted polyamines had been reported. This was the case even though the above symmetrical bis(alkyl)polyamines, such as BENSpm and BESpm, were reported to have been synthesised, and evaluated successfully for anti-tumor activity. In whole, the synthesis of these analogues are quite complex. It needs the selective protection and deprotection of the internal and external nitrogens .
The structures for unsymmetrically substituted alkyl-polyamine analogues are given in Figure 8.
2. Parasetic Diseases
There are more than 45 000 identified species of protozoa. Almost 10 000 are parasitic in invertebrates, and in each species of vertebrate . Therefore, it’s not surprising that humans and animals acts as hosts to protozoa. However the diseases caused are excessive to the number of species involved. The protozoa infecting humans ranges from types which are never pathogenic, to types belonging to the order of kinetoplastida which causes the major mammalian diseases:
- African trypanosome – i.e. Trypanosome brucei, causes sleeping sickness.
- South American trypanosomes – i.e. Trypanosome Cruzi, causes Chaga’s disease.
- Leishmania parasites cause different kind of diseases (Leishmaniasis) .
2.1 African Trypanosome
2.1.3 Life Cycle
Establishment of the infective stage of the protozoan into the human host occurs with the bite of an infected tsetse fly vector belonging to family of Glossina. In preparing for its bloody meal, the insects secrete parasite-laden saliva (spit) into the dermis of its victim. This opens the blood vessels and stops the coagulation of the blood. This allow infection by the metacyclic trypomastigotes of the protozoan .
Figure 12. Life cycle of Trypanosoma brucei
The diseases caused by the two organisms are comparable except for their internal development in humans. In general, a short period after the introduction of metacyclic trypomastigotes through the bite of the tsetse fly, an inflammatory reaction of 1 to 2 days duration takes place at the site of the bite . The characteristic reaction, a trypanosomal chancre (Fig 13 A),comes with reddening of the skin, a swelling of 1 to 5 cm diameter, and enlargement of the opposite lymph nodes.
Characteristically, the diagnosis of the disease is a multistep procedure :
1) Clinical assessment, precisely when there are easy to see neurological signs and/or mental dullness which occur when there enlarged and sensitive cervical lymph nodes known as Winterbottom’s sign (Fig 13 B)
2) Examination of the bloody smears, marrow, or cerebrospinal fluid for trypomastigotes.
3) At last, if results of the previous two steps are unconvincing, the blood is tested for antibodies.
2.2 American Trypanosomiasis (Trypanosoma Cruzi)
From the southern part of the United States, through Mexico and Central America and in South America as far south as Argentina, there are rodents, opossums and armadillos that are infected with a trypanosome and are capable of causing disease in humans .
2.2.3 Life cycle
The metacyclic trypomastigotes of T. Cruzi grow in hindgut of the cone nosed in triatoma infestans and a lot of related hemipteran insects see (Fig. 15). Because growth to infective phase takes place in the hindgut rather than the silvary glands, T. cruzi is placed in the section Stercoraria . Metacyclic forms are excreted with the feces of the bug. Normally this occurs when it is taking a blood meal from a vertebrate host. The infection is caused by the infected fecal material is rubbed into bite wound, eyes, or mucous membranes.
As soon as introduction into the human takes place the parasite attacks macrophages of the subcutaneous tissue at the point of infection. This causes a local oedematous swelling named a chagoma [22.21]. Human patients regularly show signs of oedematous patches over the body, which occur mainly on one side of the face. This one-sided oedema is frequently peri-orbital and linked with conjunctivitis. The condition is known as Romaña’s sign (see fig. 18). In early stages of the disease, parasites flourish in infected tissues and also in the circulating blood. As the infection becomes more chronic, the number of parasites in the circulation decreases significantly until they are almost undetectable.
In acute cases, 1 to 3 weeks after infection, fever, headache, malaise and prostration can still occur. Enlargement of the liver and spleen and also myocardial damage could follow. But cardiac participation and gastrointestinal symptoms (mega-syndrome) may not be obvious until several years after the primary infection. [19, 20]
2.3.1 Cutaneous Leishmaniasis
Oriental sore, as observed in the old World, is caused by leishmanias belonging to the Leishmania tropica complex. There are three serologically and biochemically separate species. All are transmitted by sandflies belonging to the genus phlebotomus . L. topica generates chronic disease that when not treated, lasts for several years or longer. It is diagnosed when dry lesions form that only ulcerate after a number of months. They normally occur singlely and usually on the face.
2.3.2 Mucocutaneous Leishmaniasis
In Latin America, an infection due to the Leishmania braziliensis has been observed . The outstanding feature of this disease is the progression in some patients to ulcers in the oral or nasal mucosa. In Brazil this disease is known as espundia. The rate of mucous membrane participation for the endemic region as whole is significantly lower.
2.3.3 Visceral Leishmaniasis
Visceral Leishmaniasis is generally known by its Indian name of Kala-azar. It is not anymore thought to be caused by a single agent. but of at least three species belonging to the donovani complex. However they are clinically and biochemically dissimilar and have different geographic distributions. As in cutaneous and mucocutaneous Leishmaniasis, the contributing organisms are parasites of the reticuloendothelial system. Similar to those discussed before, the parasites causing the Kala-azar are not restricted to the reticuloendothelial cells of the subcutaneous tissues and mucous membranes but could be established within the body .
2.3.4 The life cycle
2.4.1 New drugs
3.1 Method designed through the retrosynthetic analysis
3.1.1 Stage 1 small scale: synthesis of cyanoethylpropan-1, 3-diamine
A preliminary small scale experiment was attempted. When all the starting materials [list names and quantities] were added together and left to stir over 1 hour, the reaction mixture turned into white solid. This was due to 4-bromobutyronitrile [corrected now] been added too quickly in one portion, rather it than adding drop wise over 1 hour. Also the mole ratios were not correct. The experiment was repeated but this time on large scale with the correct mole ratios and adding 4-bromobutyronitrile drop wise.
3.1.2 Stage 1 repeated on large scale synthesis
The TLC run of samples taken from the reaction did not show amine spot that would indicate that the reaction had occurred. However NMR analysis showed that starting material 4-bromobutyronitrile had been regenerated. This was determined by comparing the NMR spectrum of 4-bromobutyronitrile and the NMR spectrum of the reaction mixture alongside each other. Therefore it was possible to determine whether the reaction has occurred or one of the starting materials had been regenerated. The conclusion was that the expected reaction at N8 carbon had not occurred. At this point, this method was abandoned and was necessary to refer to previous literature methods for the synthesis of cyanoethylpropan-1, 3-diamine.
4. Stage 1 repeated with previous method by Zhang et al 
4.1 synthesis of 3-[(3-cyanopropyl) amino] propylamine
5. Stage 2 was carried with previous method by Humora and Quick . [when there are only 2 authors write both names]
5.1 Synthesis of 1HNMR of NN-di-tert-butoxycarbonyl-N-(3-cyanopropyl)-1, 3-diamine.
Product prepared was a orange crystals of 1,3-diamine salt, rather than the 1,4-diamine oil prepared by Humora and Quickl .
Mass of product = 0.70 g
6. Stage 1 IR interpretations
6.1 Large scale synthesis of 3-[(3-cyanopropyl)amino]propylamine based on the method by Zhang et al .
IR of fraction 1
IR of fraction 2
IR of fraction 3
IR of fraction 4
7. Stage 1 NMR interpretations
1H NMR of fraction 1
C13 NMR of fraction
Assigned peaks (1,2, 3 etc) to hydrogens on structure of fraction 1
Assigned peaks (a,b, c etc) to carbons on structure of fraction 1
1 2 3 4
a b c d
1H NMR of fraction 2: unreacted amine [use lower initial letter for table data]
1H NMR of fraction 2 + D2O
C13 NMR of fraction 2
13 NMR of fraction 2
Assign peak (1,2, 3 etc) to hydrogens on structure of fraction 2
Assign peak (a,b, c etc) to carbons on structure of fraction 2
1H NMR of fraction 3
1H NMR of fraction 3 + D2O shake
C13 NMR of fraction 3
Assign peak (1,2, 3 etc) to hydrogens on structure of fraction 3
Assign peak (a,b, c etc) to carbons on structure of fraction 3
1H NMR of fraction 4
C13 NMR of fraction 4
Assign peak (1,2, 3 etc) to hydrogens on structure of fraction 4
Assign peak (a,b, c etc) to carbons on structure of fraction 4
Thin layer Chromatography (Tlc) of fraction 4
8. Stage 2 IR and NMR interpretations
IR of NN-di-tert-butoxycarbonyl-N-(3-cyanopropyl)-1, 3-diamine
1H NMR of NN-di-tert-butoxycarbonyl-N-(3-cyanopropyl)-1, 3-diamine
C13 NMR of NN-di-tert-butoxycarbonyl-N-(3-cyanopropyl)-1, 3-diamine
Assign peak (1,2, 3 etc) to hydrogens on structure of stage 2
Assign peak (a,b, c etc) to carbons on structure of stage 2