Polyamines. Illustrate your essay with specific examples

diaminopropyl-tetramethylenediamine]

These polyamines are derived from ornithine following an initial
decarboxylation
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) ^[5]

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⁺

1.6.1 Apoptotic Cell Death^[15]

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^[15]

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). ^[13] DFMO has been used many times in the last 30-40 years for research:

1. to elucidate the polyamine synthesis and pathways of catabolism.
2. the assessment of the polyamine controlled mechanisms linking cell proliferation and differentiation.
3. the inhibition of cancer cell growth.
4. the improvement of human cancer therapies and also in cancer prevention programs.
5. the development of new cancer chemotherapies
6. 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. ^[13]

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
cells. ^[4]
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. ^[12]

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:

1. changing the chain-length of methylene groups that bridge the primary and secondary amino groups.
2. adding substituents at different positions.
3. alkylating the amino end groups.

Bis(ethyl) derivatives of putrescine or spermidine and spermine were synthesised by porter et al ^[17]. 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 ^[18].
The structures for unsymmetrically substituted alkyl-polyamine analogues are given in Figure 8.

2.1.1 Distribution

2.1.2 Vector

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 [21].
Figure 12. Life cycle of Trypanosoma brucei^[24]

2.1.4 Diagnosis

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 ^[21]. 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 ^[22]:

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.

2.2.1 Distribution

2.2.2 Vector

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 ^[19]. 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.

2.2.4 Diagnosis

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 ^[24]. 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 ^[19]. 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 ^[22].

2.3.4 The life cycle

2.4.1 New drugs

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.