Yerevan State Medical University After M. Heratsi

Home , Benzobarbital, Mesocarb, Picamilon, Semax, Sleep induction

YEREVAN STATE MEDICAL UNIVERSITY AFTER M. HERATSI

DEPARTMENT OF PHARMACY

Balasanyan M.G. Zhamharyan A.G. Afrikyan Sh. G. Khachaturyan M.S. Manjikyan A.P.

MEDICINAL CHEMISTRY

HANDOUT for the 3-rd-year pharmacy students (part 2)

YEREVAN 2017

Analgesic Agents

Agents that decrease pain are referred to as analgesics or as analgesics. Pain relieving agents are also called antinociceptives. An analgesic may be defined as a drug bringing about insensibility to pain without loss of consciousness. Pain has been classified into the following types: physiological, inflammatory, and neuropathic. Clearly, these all require different approaches to pain management. The three major classes of drugs used to manage pain are opioids, nonsteroidal anti-inflammatory agents, and non opioids with the central analgetic activity.

Narcotic analgetics

The prototype of opioids is Morphine. Morphine is obtained from opium, which is the partly dried latex from incised unripe capsules of Papaver somniferum. The opium contains a complex mixture of over 20 alkaloids. Two basic types of structures are recognized among the opium alkaloids, the phenanthrene (morphine) type and the benzylisoquinoline (papaverine) type (see structures), of which morphine, codeine, noscapine (narcotine), and papaverine are therapeutically the most important.

The principle alkaloid in the mixture, and the one responsible for analgesic activity, is morphine. Morphine is an extremely complex molecule. In view of establish the structure a complicated molecule was to degrade the: compound into simpler molecules that were already known and could be identified. For example, the degradation of morphine with strong base produced methylamine, which established that there was an N-CH3 fragment in the molecule. Once a structure had been proposed, chemists would then attempt to synthesize it. If the properties of the synthesized compound were the same as those of the natural compound, then the structure was proven. At 1925 Sir Robert Robinson proposed the correct structure of morphine. A full synthesis was

2 achieved in 1952, and the structure proposed by Robinson was finally proved by X-ray crystallography in 1968 (164 years after the original isolation).

The molecule contains five rings numbered A-E and has a pronounced T shape. The principally important groups for SAR study are:  The phenolic OH  The 6-alcohol  The double bond at 7-8  The N-methyl group  The ether bridge

The phenolic OH. Methylating the phenolic OH causes a drastic drop in receptor binding. Codeine is the methyl ether of morphine. The binding affinity for codeine is only 0.1% that of morphine. This drop in affinity is observed in other analogues containing a masked phenolic group, so a free phenolic group is crucial for analgesic activity. Nevertheless, if codeine is administered to patients, its analgesic effect is 20% that of morphine, because codeine is metabolized by O-demethylation in the liver to give morphine. Thus, codeine can be viewed as a prodrug for morphine.

The 6-alcohol. Masking or losing the alcohol group does not decrease analgesic activity, but it improves activity as a result of improved pharmacokinetic properties. In other words, the drugs concerned reach the target receptor more easily. In this case, the morphine analogues shown are less polar, than morphine because a polar alcohol group has been masked or lost. As a result they cross the blood-brain barrier into the 3 central nervous system more efficiently and accumulate at the target receptors in greater concentrations; hence the better analgesic activity.

For example to compare the activities of morphine, 6-acetylmorphine and diamorphine (heroin) demonstrate that the most active (and the most dangerous) compound of the three is 6-acetylmorphine, which is four times more active than morphine. Heroin is also more active than morphine by a factor of two, but less active than 6-acetylmorphine. 6-acetylmorphine is less polar than morphine and will enter the brain more quickly and in greater concentrations. The phenolic group is free and therefore it will interact immediately with the analgesic receptors.

Heroin has two polar groups which are masked and is therefore the most efficient compound of the three in crossing the blood-brain barrier. Before it can act at the receptor, however, the 3-acetyl group has to be removed by esterases in the brain. This means that it is more powerful, than morphine because of the ease with which it crosses the blood-brain barrier, but less powerful than 6-acetylmorphine since the 3-acetyl group has to be hydrolyzed. Heroin and 6-acetylmorphine are both more potent analgesics, than morphine. Unfortunately, they also have greater side effects as well as severe tolerance and dependence characteristics. Heroin is still used to treat terminally ill patients suffering chronic pain, but 6-acetylmorphine is so dangerous that its synthesis is banned in many countries.

To conclude, the 6-hydroxyl group is not required for analgesic activity, and its removal can be beneficial to analgesic activity, and improves the pharmacokinetic properties of drugs.

The double bond at 7-8. Several analogues have shown that the double bond is not necessary for analgesic activity, for example dihydromorphine.

The N-methyl group. The introduction of charge destroys analgesic activity. The N- oxide and the N-methyl quaternary salt of morphine are both inactive. No analgesia is observed, as a charged molecule has very little chance of crossing the blood-brain barrier and reaching it target receptor. If these same compounds are injected directly into the brain (or in vitro), both compounds actually have a similar analgesic activity to morphine. It demonstrates, that the nitrogen atom of morphine must be ionized, when it binds to the receptor. N-demethylation to give normorphine reduces activity, but does not eliminate it. The secondary NH group is more polar, than the original tertiary group and so normorphine is less efficient at crossing the blood-brain barrier, leading to a drop in activity. The fact that significant activity is retained shows that the methyl substituent is not essential to activity.

X = NH Normorphine

X = N-Oxide

The nitrogen itself is crucial, however. If it is

X= Quaternary replaced by carbon, all analgesic activity is lost. To conclude, the nitrogen atom is essential

5 to analgesic activity and interacts with the analgesic receptor in the ionized form.

The aromatic ring. The aromatic ring is essential. Compounds lacking it show no analgesic activity.

The ether bridge. The ether bridge is not required for analgesic activity.

Stereochemistry. Morphine is an asymmetric molecule containing several asymmetric centers, and exists naturally as a single stereoisomer. When morphine was first synthesized, it was made as a racemic mixture of the naturally occurring stereoisomer plus its mirror image. These were separated and the unnatural mirror image was tested for analgesic activity.

It turned out to have no activity, because the pharmacological activity of morphine is realized by the interaction between morphine and its receptor. As we discussed, there are at least three important interactions involving the phenol, the aromatic ring, and the amine.

The receptor has complementary binding groups placed in such a way that they can interact with all three groups. The mirror image morphine, on the other hand, can interact with only one binding region at anyone time.

Epimerization of a single asymmetric centre such as the 14-position is not beneficial either, since changing the stereochemistry at even one asymmetric centre can result in a drastic change of shape, making it difficult for the molecule to bind. To sum up, the important functional groups for analgesic activity in morphine are the phenol, aromatic ring and amine. The stereochemistry of the molecule is crucial in ensuring that these groups are in the correct position for binding. KEY POINTS

• Morphine is extracted from opium and is one of the oldest drugs used in medicine. • Morphine is a powerful analgesic, but has various side effects, the most serious being respiratory depression, tolerance, and dependence. • The structure of morphine consists of five rings forming a T-shaped molecule. • The important binding groups on morphine are the phenol, the aromatic ring, and the ionized amine. • Analgesic activity of morphine analogues requires the presence of the important binding groups as well as the ability to cross the blood-brain barrier. • Various opiates such as codeine and diamorphine (heroin) act as prodrugs for morphine. Synthetic analogs of Morphine

Morphine is still one of the most effective painkillers available to medicine. Unfortunately, it has a large number of side effects, which include: depression of the respiratory centre, constipation, excitation, euphoria, nausea, pupil constriction, tolerance and dependence. Considering the problems associated with morphine, there is a need for novel analgesic agents, which retain the analgesic activity of morphine, but which have fewer side effects and can be administered orally. The follow classical drug design strategies were used in obtaining novel analgesic structures:

 Drug extension 7

 Simplification or drug dissection  Rigidifitation

The strategy of drug extension involves the addition of extra functional groups to a lead compound in order to probe for extra binding regions in a binding site. Many analogues of morphine containing extra functional groups have been prepared. These have rarely shown any improvement. There are two exceptions, however. The introduction of a hydroxyl group at position 14 increases activity and suggests that there might be an extra hydrogen bond interaction taking place with the binding site.

The other exception involves the variation of alkyl substituent on the nitrogen atom. As the alkyl group is increased in size from a methyl to a butyl group, the activity drops to zero. With a larger group such as a pentyl or a hexyl group, activity recovers slightly. None of this is particularly exciting, but when a phenethyl group is attached, the activity increases l4-fold relative to morphine - a strong indication that a hydrophobic binding region has been located which interacts favorably with the new aromatic ring.

To conclude, the size and nature of the group on nitrogen is important to the activity spectrum. Drug extension can lead to better binding by making use of additional binding interactions. It was occurred important results when an allyl or a cyclopropylmethylene group is attached to nitrogen and was synthesized tree analogues - Naloxone, Naltrexone and Nalorphine.

Naloxone and naltrexone have no analgesic activity at all, and they prevent the analgesic action of morphine, that is they bind to the analgesic receptors without switching them on. Once they have bound to the receptors, they block morphine from binding. The fact that morphine is blocked from all its receptors means that none of its side effects are produced either, and it is the blocking of these effects that makes antagonists extremely useful. Naltrexone is eight times more active, than naloxone as an antagonist and is given to drug addicts who have been weaned off morphine or heroin. Nalorphine is a strong antagonist and blocks morphine from its receptors. Therefore, no analgesic activity should be observed. But a very weak analgesic activity is observed (agonist-antagonist) and what is more, this activity appears to be free of the undesired side effects. This was the first sign that a non addictive, safe analgesic might be possible. The fact of existence a compound, which acts both as an antagonist, and as an agonist and produce analgesia with the so weak activity and free of the side effects, indicates, that there are several types of analgesic receptor rather than just one. It was proved, that analgesic receptors is multiple receptors and there are at least three types of analgesic receptor. Morphine activates them all. Nalorphine binds to all three types of analgesic receptor and blocks morphine from all three. It acts as a true antagonist at two of the receptors, but at the third it acts as a weak or partial agonist. The third receptor is controlling something like an ion channel. Morphine is a strong agonist and interacts strongly with this receptor, leading to a change in receptor conformation which fully opens the ion channel. Naloxone is a pure antagonist. It binds strongly, but does not produce the correct change in the receptor conformation. Therefore, the ion channel remains closed. Nalorphine binds to the third receptor and changes the tertiary structure of the receptor very slightly, leading to a slight opening of the ion channel. It is therefore a weak agonist at this receptor, but it is also an antagonist since it blocks morphine from switching the receptor on fully. The results observed with nalorphine show that activation of this third type of analgesic receptor leads to analgesia without the undesirable side effects associated with the other two analgesic receptors. 9

Unfortunately, nalorphine has hallucinogenic side effects resulting from the activation of a non analgesic receptor, and it is therefore unsuitable as an analgesic, but for the first time a certain amount of analgesia had been obtained without the side effects of respiratory depression and tolerance.

The strategy of simplification or drug dissection based on removing of some rings from morphine.

1. Removing ring E. Removing ring E leads to complete loss of activity. This emphasizes the importance of the basic nitrogen to analgesic activity.

2. Removing ring D.

Removing the oxygen bridge gives a series of compounds called the morphinans, which have useful analgesic activity. This demonstrates that the oxygen bridge is not essential. N-Methylmorphinan was the first such compound tested, and is only 20% as active as morphine, but since the phenolic group is missing, this is not surprising. The more 10 relevant levorphanol structure is five times more active than morphine and, although side effects are also increased. Levorphanol has a massive advantage over morphine in that it can be taken orally and lasts much longer in the body. This is because levorphanol is not metabolized in the liver to the same extent as morphine. The same strategy of drug extension already described for the morphine structures was tried on the morphinans with similar results. For example, adding an allyl substituent on the nitrogen gives antagonists. Adding a phenethyl group to the nitrogen greatly increases potency. Adding a 14-hydroxyl group also increases activity.

KEY POINTS  Morphinans are more potent and longer acting, than their morphine counterparts, but they also have higher toxicity and comparable dependence characteristics.  Modifications carried out on the morphinans have the same biological results as they do with morphine. This implies that morphine and morphinans are binding to the same receptors in the same way.  The morphinans are easier to synthesize since they are simpler molecules. 3. Removing rings C and D. Opening both rings C and D gives an interesting group of compounds called the benzomorphans, which retain analgesic activity. One of the simplest of these structures is metazocine, which has the same analgesic activity as morphine. Notice that the two methyl groups in metazocine are cis with respect to each other and represent the remnants of the C ring. The same chemical modifications carried out on the benzomorphans as described for the morphinans and morphine, produce the same biological effects, implying a similar interaction with the analgesic receptors. For example, replacing the N-methyl group of metazocine with a phenethyl group gives phenazocine, which is four times more active than morphine and is the first compound to have a useful level of analgesia without dependence properties.

Further developments led to pentazocine, which has proved to be a useful short-term analgesic with a very low risk of addiction. A newer compound (bremazocine) has a longer duration, has 200 times the activity of morphine, appears to have no addictive properties, and does not depress breathing. These compounds appear to be similar in their action to nalorphine in that they act as antagonists at two of the three types of analgesic receptors, but act as agonists at the third. The big difference between nalorphine and compounds like pentazocine is that the latter are far stronger agonists, resulting in a more useful level of analgesia.

Unfortunately, many of these compounds have hallucinogenic side effects due to interactions with a non analgesic receptor. KEY POINTS • Rings C and D are not essential to analgesic activity. • Analgesia and addiction are not necessarily co-existent. • 6,7-Benzomorphans are clinically useful compounds with reasonable analgesic activity, less addictive liability, and less tolerance. • Benzomorphans are simpler to synthesize, than morphine and morphinans. 4. Removing rings B, C, and D. Removing rings B, C, and D gives a series of compounds known as 4- phenylpiperidines. Pethidine (meperidine) is a weaker analgesic, than morphine, but shares the same undesirable side effects. On the plus side, it has a rapid onset and a shorter duration of action. As a result, it has been used as an analgesic in childbirth.

The rapid onset and short duration of action mean that there is less chance of the drug depressing the baby's breathing once it is born.

Activity can be increased six-fold by introducing the phenolic group and altering the ester to a ketone to give ketobemidone.

There is some doubt as to whether the piperidines act in the same way as morphine at analgesic receptors, since some of the chemical adaptations for example, adding allyl or cyclopropyl groups does not give antagonists and more likely, that piperidines bind with receptors in different ways. One of the most successful piperidine derivatives is fentanyl, which is up to 100 times more active than morphine. The drug lacks a phenolic group, but is very lipophilic. As a result, it can cross the blood-brain barrier efficiently.

KEY POINTS • Rings C, D, and E are not essential for analgesic activity. • Piperidines retain side effects, such as addiction and depression of the respiratory centre. • Piperidine analgesics are faster acting and have shorter duration. 13

• The quaternary centre present in piperidines is usually necessary (fentanyl is an exception). • The aromatic ring and basic nitrogen are essential to activity, but the phenol group is not. • Piperidine analgesics appear to bind with analgesic receptors in a different manner to previous groups. 5. Removing rings B, C, D and E. The analgesic methadone was discovered and has proved to be a useful agent, comparable in activity to morphine. It is orally active and has less severe emetic and constipation effects. Side effects such as sedation, euphoria, and withdrawal symptoms are also less severe and therefore the compound has been given to drug addicts as a substitute for morphine or heroin in order to wean them off these drugs. This is not a complete cure, as it merely swaps an addiction to heroin or morphine for an addiction to methadone. This is considered less dangerous, however. The molecule has a single asymmetric centre and when the molecule is drawn in the same manner as morphine, we would expect the R-enantiomer to be the more active enantiomer. This proves to be the case, with the R-enantiomer being twice as powerful as morphine, whereas the S-enantiomer is inactive. This is quite a dramatic difference. Since the R- and S-enantiomers have identical physical properties and lipid solubility, they should both reach analgesic receptors to the same extent, and so the difference in activity is most probably due to receptor-ligand interactions.

It was synthesized a hybrid molecule involving a 4-phenylpiperidine and a methadone-like structure - Loperamide is a successful antidiarrhoeal agent which is marketed as Imodium. It is an example, when is possible to take advantage of a side effect of opiate analgesics - constipation, original side effect becomes the predominant feature. The compound is lipophilic, slowly absorbed, and prone to metabolism. It is also free from any euphoric effect, since it cannot cross the blood brain barrier. All these features make it a safe medicine, free from the addictive properties of the opiate analgesics.

The strategy of regidification is used to limit the number of conformations that a molecule can adopt. The aim is to retain the active conformation for the desired target and eliminate alternative conformations that might fit different targets. This should increase activity, improve selectivity and decrease side effects. The best examples of this tactic in the analgesic field are the oripavines, which was synthesized from thebaine (alkaloid derived from opium, which has weak expressed analgetic activity). A comparison of these structures with morphine shows that an extra ring sticks out from what used to be the crossbar of the T-shaped morphine.

Some remarkably powerful oripavines have been obtained. Etorphine, for example, is 10 000 times more potent than morphine. This is a combination of the fact that it is a very hydrophobic molecule and can cross the blood-brain barrier 300 times more easily than morphine, as well as having 20 times more affinity for the analgesic receptor site due to better binding interactions. At slightly higher doses than those required for analgesia, it can act as a knock-out drug or sedative. It has a considerable margin of safety and is used to immobilize large animals such as elephants. Adding a cyclopropyl group gives a very powerful antagonist called diprenorphine, which is 100 times more potent than nalorphine and can be used to reverse the immobilizing effects of etorphine. Diprenorphine has no analgesic activity.

A similar compound is buprenorphine, which has similar properties to drugs like nalorphine and pentazocine, in that it has analgesic activity with a very low risk of addiction. It is 100 times more active, than morphine as an agonist and 4 times more active, than nalorphine as an antagonist. It is a particularly safe drug, since it has very little effect on respiration and what little effect it does have actually decreases at high doses. Therefore, the risks of suffocation from a drug overdose are much smaller, than with morphine. The properties of buprenorphine appear to be related to its slow onset of action and slow departure from receptors. Since buprenorphine is the most lipophilic compound in the oripavine series of compounds and enters the brain very easily, the slow onset has nothing to do with how easily it reaches the receptor. Instead, it is a result of slow binding then slow departure from the binding site. Effects are gradual, so there are no sudden changes in transmitter levels. Although buprenorphine binds slowly, it binds very strongly once it is bound. As a result, less buprenorphine than morphine is required to interact with a certain percentage of analgesic receptors. On the other hand, buprenorphine is only a partial agonist and is less efficient at switching the analgesic receptors on. This means that it is unable to reach the maximum level of analgesia, which can be produced by morphine. Overall, buprenorphine's stronger affinity for analgesic receptors outweighs its relatively weak analgesic action, such that buprenorphine can produce analgesia at lower doses, than morphine. KEY POINTS

• The addition of a 14-hydroxyl group or an N-phenethyl group usually increases activity as a result of interactions with extra binding regions. • N-Alkylated analogues of morphine are easily synthesized by demethylating morphine to normorphine, then alkylating with alkyl halides. • The addition of suitable N-substituents results in compounds, which act as antagonists or partial agonists. Such compounds can be used as antidotes to morphine overdose, as treatment for addiction or as safer analgesics. • The morphinans and benzomorphans are analgesics, which have a simpler structure, than morphine and interact with analgesic receptors in a similar fashion. • The 4-phenylpiperidines are a group of analgesic compounds, which contain the analgesic pharmacophore present in morphine. They may bind to analgesic receptors slightly differently from analgesics of more complex structure. • Methadone is a synthetic agent, which contains part of the analgesic pharmacophore present in morphine. It is administered to drug addicts to wean them off heroin. • Thebaine is an alkaloid derived from opium, which lacks analgesic activity. It is the starting material for a two-stage synthesis of oripavines. • Oripavines are extremely potent compounds due to enhanced receptor interactions' and an increased ability to cross the blood-brain barrier. • The addition of N-cycloalkyl groups to the oripavines results in powerful antagonists or partial agonists, which can be used as antidotes, for the treatment of addiction or as safer analgesics. Receptor theory of analgesics The initial theory (Beckett-Casy receptor theory) on receptor binding assumed a single analgesic receptor; it was assumed that there was a rigid binding site into which morphine and its analogues could fit in a classic lock-and-key analogy. Based on the results already described, the following binding interactions were proposed: • a basic nitrogen (free base necessary as the analgesic has to cross the blood-brain barrier) which is ionized at physiological pH to form a positively charged group and then forms an ionic bond with a comparable anionic group in the receptor binding site. • The aromatic ring in morphine has to be properly orientated with respect to the nitrogen atom to allow a van der Waals interaction with a suitable hydrophobic location in the binding site. • The phenol group is probably hydrogen bonded to a suitable residue in the receptor binding site.

• There might be a 'hollow' just large enough for the ethylene bridge of C-15 and C- 16 to fit. Such a fit would help to align the molecule and enhance the overall fit.

Synthesis of morphine analogues with the different types of activity and side effects indicate on multiple analgesic receptors. It is now known at least four different receptors with which morphine can interact. Three of these promote analgesia: mu (), kappa (K) and delta (δ). A fourth receptor a completely different non-analgesic receptor called the sigma σ and associated with the hallucinogenic activity. It was establish the new class of opioid receptors orphan opioid receptor, which did not bind the classical opioid peptide and caused hyperalgesia (nociception), its endogenous ligand is nociceptin.

All the opioid receptors are Gi-dependent. The μ-recptor is responsible for the opening of a potassium ion channel.

The kappa-receptor is responsible for analgesia and sedation, and lacks serious side effects. Receptor-ligand binding results in the closure of a calcium ion channel.

The δ -receptor is favored by the enkephalins. Receptor ligand binding results in the activation and fragmentation of a Gi-protein. The resulting α1-suburnt inhibits the enzyme adenylate cyclase and prevents the synthesis of cAMP.

Relative activities of analgesics

Morphine itself is not present in humans and therefore the body must be using a different chemical as its endogenous painkiller. At least 15 endogenous peptides have now been discovered (the enkephalins, dynorphins, and the endorphins), varying in length from 5 to 33 amino acids. These compounds are thought to be neurotransmitters or neurohormones in the brain and operate as the body's natural painkillers, as well as having a number of other roles. It is known that enkephalins show preference for the - δ receptor, whereas dynorphins show selectivity for the kappa-receptor, and β-endorphins show selectivity to both the δ and μ-receptors. The most recently discovered opioid peptides are the endomorphins. These peptides have a strong preference for the μ - receptor.

Beyond the important binding groups for each receptor - phenol, the aromatic ring, and the ionized nitrogen centre there are subtle differences between the receptors. As a result, some analgesics show preference for one analgesic receptor over another or interact in different ways. 19

It is suggested that there are two accessory hydrophobic binding regions present in an analgesic receptor. The agonist binding region is further away from the nitrogen and positioned axially with respect to it. The antagonist region is closer and positioned equatorially. If the substituent is phenethyl group, it’s in the axial position; the aromatic ring is in the correct position to interact with the accessory agonist binding region. If the phenethyl group is in the equatorial position, the aromatic ring is placed beyond the antagonist binding region and cannot bind to either of the accessory regions. The overall result is that the molecule binds with the phenethyl group axial, resulting in increased agonist activity. It is proposed, that a molecule such as phenazocine, a phenethyl group acts as an agonist, because it only binds to the agonist binding region.

If the phenethyl group replaced by an allyl group we have partial agonist, because in the equatorial position, the allyl group binds strongly to the antagonist binding region, whereas in the axial position it barely reaches the agonist binding region, resulting in a weak interaction. It is proposed, that a molecule such as nalorphine with an allyl group can bind both agonist and antagonist regions and therefore acts as an agonist at one receptor and an antagonist at another. The ratio of these effects would depend on the equilibrium ratio of the axial and equatorial substituted isomers.

A compound which is a pure antagonist would be forced to have a suitable substituent in the equatorial position. It is believed that the presence of a 14-hydroxyl group sterically hinders the isomer with the axial substituent, and forces the substituent to remain equatorial.

KEY POINTS • The Beckett-Casy receptor theory identifies the importance of a hydrogen bonding interaction via a phenol group, an ionic interaction via a charged amine, and van der Waals interactions involving the aromatic ring. Modifications to the theory can account for extra binding interactions as a result of drug extension. • There are three different analgesic receptors (μ, K, and δ). All require the presence of a pharmacophore involving the phenol, aromatic ring, and ionized amine. • The μ-receptor is responsible for the opening of a potassium ion channel. Morphine binds most strongly to the μ-receptor. This receptor is responsible for the serious side effects associated with morphine. • The K-receptor is responsible for analgesia and sedation, and lacks serious side effects. Receptor-ligand binding results in the closure of a calcium ion channel. • The δ-receptor is favored by the enkephalins. Receptor ligand binding results in the activation and fragmentation of a Gj-protein. The resulting α1-suburnt inhibits the enzyme adenylate cyclase and prevents the synthesis of cAMP. • It is proposed that there are two accessory hydrophobic binding regions in the receptor binding site. An agent will act as an agonist or antagonist depending on which of these regions it can access. • Enkephalins, dynorphins, endomorphins and endorphins are peptides, which act as the body's natural painkillers. The presence of an N-terminal tyrosine is crucial to activity.

• Analogues of enkephalins have been designed to be more stable to peptidases, by the inclusion of unnatural amino acids, d-amino acids or N- methylated peptide links. • The -receptor is responsible for the hallucinogenic properties of some analgesics.

Non steroidal anti-inflammatory drugs (NSAIDs)

Prostaglandins, Thromboxanes, Prostacyclin and Leukotrienes

Prostaglandins are naturally occurring 2O-carbon cyclopentano-fatty acid derivatives produced in mammalian tissue from polyunsaturated fatty acids. They belong to the class of eicosanoids, derived from membrane phospholipids. The eicosanoids are derived from unsaturated fatty acids and include the following groups of compounds: prostaglandins, thromboxanes, prostacyclin and leukotrienes. They have been found in essentially every compartment of the body, beside erythrocytes.

All naturally occurring prostaglandins (PGs) possess this substitution pattern, a 15- hydroxy group and a trans double bond at C-13. Unless a double bond occurs at the C- 8- C-12 positions, the two side chains (the carboxyl-bearing chain termed the α-chain and the hydroxyl-bearing chain termed the β-chain) are of the trans stereochemistry depicted. The PGs are classified by the capital letters A, B, C, D, E, F, G, H and I depending on the nature and stereochemistry of oxygen substituents at the 9-and 11- positions. For example, members of the PGE series possess a keto function at C-9 and a hydroxyl group at C-11, whereas members of the PGF series possess a hydroxyl groups at both of these positions. The number of double bonds in the side chains connected to the cyclopentane ring is designated by subscripts 1, 2 or 3, indicative of the nature of the fatty acid precursor, e.g. PGE1, PGE3. There are α and β types: PGF1β,

PGF2α, depending on hydroxyl groups’ orientation in the cyclopentane ring.

Prostaglandins are derived biosynthetically from unsaturated fatty acid precursors. The number of double bonds contained in the naturally occurring PGs reflects the nature of the biosynthetic precursors. Those containing one double bond are derived from 8,11,14-eicosatrienoic acid, those with two double bonds from arachidonic acid (5,8,11,14-eicosatetraenoic acid), and those with three double bonds from 5,8,11,14,17- eicosapentenoic acid. The most common of these fatty acids in humans is arachidonic acid and hence PGs of the 2 series play an important biological role. Arachidonic acid is derived from dietary linoleic acid or is ingested from the diet and esterified to phospholipids (primarily phosphatidyIethanolamine or phosphatidylcholine) in cell membranes. Various initiating factors (interleukin 1, leucotrane) interact with membrane receptors coupled to G proteins (guanine nucleotide-binding regulatory proteins) activating phospholipase A2 which, in turn, hydrolyzes membrane phospholipids resulting in the release of arachidonic acid. Other phospholipases (e.g., phospholipase C) are also involved. Phospholipase C differs from phospholipase A2 by inducing the formation of 1,2-diglycerides from phospholipids with the subsequent release of arachidonic acid by the actions of mono- and diglyceride lipases on the diglyceride. A polypeptide produced by leukocytes, leukotrienes, interleukin-1, which mediates inflammation, increases phospholipase activity and thus PG biosynthesis. The steroidal anti-inflammatory agents (corticosteroids) appear to act, in part, by inhibiting these phospholipases. The liberated arachidonic acid may then be acted on by two major enzyme systems: arachidonic acid cyclooxygenase (prostaglandin endoperoxide synthetase or COX) to produce prostaglandins, thromboxanes and prostacyclin, or by lipoxygenases to produce Ieukotrienes. Interaction of arachidonic acid with COX in the presence of oxygen and heme produces first the cyclic endoperoxides, PGG2 and thence, through its peroxidase activity, to PGH2, both of which are chemically unstable and decompose rapidly. PGE2 is formed by the action of PGE isomerase and PGD2 by the actions of isomerases or glutathione-S-transferase on PGH2 while PGF2a is formed from PGH2 via an endoperoxide reductase system. It is at the cyclooxygenase step at which the NSAIDs inhibit PG biosynthesis preventing inflammation.

Biosynthesis of prostaglandins from arachidonic acid.

COX was first purified in 1976 and first cloned in 1988. Among the more significant advances of the past decade was the isolation of a second form of the COX enzyme, named COX-2, whose expression is inducible by cytokines and growth factors. COX-1 and COX-2 are very similar in structure and are almost identical in length varying from 599 (human) to 602 (mice) amino acids in COX-1 and 603 (mice) to 604 (human) for COX-2. Both isoforms possess molecular masses of 70-74 Kda and contain just over 600 amino acids with an approximately 60% homology within the same species. COX-2 contains an 18 amino acid insert near the C-terminal end of the enzyme that is not present in COX-1, but all other residues that have been previously identified as being essential to the catalytic activity of COX-1 are present in COX-2. The three-dimensional X-ray crystal structure of COX-2 derived from human can be superimposed on that of COX-1. Residues that form the substrate binding channel, the catalytic sites and those residues immediately adjacent are essentially identical with the exception of two minor differences. The isoleucine at positions 434 and 523 in COX-1 is exchanged for valine in COX-2. The smaller size of Val 523 in COX-2 allows inhibitor

24 access to a side pocket of the main substrate channel while the longer side chain of lIe in COX-l sterically blocks inhibitor access. A major difference between COX-1 and COX-2 is that COX-2 lacks a sequence of 17 amino acids from the N-terminus, but contains a sequence of 18 amino acids at the C terminus compared to COX-I. This difference causes a difference in the numbering systems of the two isoforms such that the serine residue acetylated by aspirin in COX-1 is numbered Ser 530, while in COX-2 the serine residue acetylated is Ser 516. Yet the, amino acid residues, which are thought to be responsible for providing the catalytic role are the same with both isoforms displaying similar ability to convert arachidonic acid to PGH2. COX-l appears to be more specific for fatty acid substrates than COX-2 since COX-2 accepts a wider range of fatty acid substrates than COX-I. COX-l primarily metabolizes arachidonic acid while COX-2 metabolizes C18 and C20 fatty acid substrates. Selective inhibitors of COX-2 do not bind to Arg 120 that is used by the -COOH of arachidonic acid and the carboxylic acid selective or nonselective COX-1 inhibitors. From a therapeutic viewpoint, the major difference between COX-1 and COX-2 lies in physiological function rather than structure. Little COX-2 is present in resting cells but its expression can be induced by cytokines in vascular smooth muscle, fibroblasts and epithelial cells leading to the suggestion that COX-l functions to produce PGs that are involved in normal cellular activity (protection of gastric mucosa, maintenance of kidney function) while COX-2 is responsible for the production of PGs at inflammatory sites. Inducible COX-2 linked to inflammatory cell types and tissues are believed to be the target enzyme in the treatment of inflammatory disorders by NSAIDs. Until recently, most NSAIDs inhibited both COX-1 and COX-2 but with varying degrees of selectivity. Selective COX-2 inhibitors may eliminate side effects associated with NSAIDs due to COX-1inhibition, such as gastric and renal effects. Prostaglandins are rapidly metabolized and inactivated by various oxidative and reductive pathways. The initial step involves rapid oxidation of the 15-OH to the corresponding ketone by the prostaglandin specific enzyme prostaglandin 15-0H dehydrogenase. This is followed by reduction of the C13, C14 double bond by prostaglandin 13-reductase to the corresponding dihydro ketone, which for PGE2 represents the major metabolite in plasma. Subsequently, enzymes normally involved in β- and ώ-oxidation of fatty acids more slowly cleave the α-chain and oxidize the C-20 terminal methyl group to the carboxylic acid derivative, respectively. Hence, dicarboxylic acid derivatives containing only 16 carbon atoms are the major metabolites of PGE1 and PGE2. Alternatively, non prostanoids can also be formed from PGH2 as illustrated in Figure. Thromboxane synthetase acts on PGH2 to produce thromboxane A2 (TxA2) while prostacyclin synthetase converts PGH2 to prostacyclin (PGI2), both of which 25 possess short biologic half-lives. TxA2, a potent vasoconstrictor and inducer of platelet aggregation, has a biologic half-life of about 30 seconds, being rapidly non enzymatically converted to the more stable, but inactive, TxB2. Prostacyclin, a potent hypotensive and inhibitor of platelet aggregation, has a half-life of about three minutes and is non enzymatically-converted to 6-keto-PGFI. Platelets contain primarily thromboxane synthetase while endothelial cells contain primarily prostacyclin synthetase. Considerable research efforts are being expended in the development of stable prostacyclin analogues and thromboxane antagonists as cardiovascular agents.

Biosynthesis of thrornboxanes, prostacyclin and leukotrienes.

The existence of distinct prostaglandin receptors may explain the broad spectrum of action displayed by the prostaglandins. The nomenclature of these receptors is based on the affinity displayed by natural prostaglandins, prostacyclin or thromboxanes at each receptor type. Thus, EP receptors are those receptors for which the PGEs have high affinity, FP receptors for PGFs, DP receptors for PGDs, IP receptors for PGI2 and TP receptors for TxA2. These receptors are coupled through G proteins to effector mechanisms that include stimulation of adenyl cyclase, and hence increased cAMP

26 levels, and phospholipase C that results in increased levels of inositoll,4,5-triphosphate. Three distinct receptors for leukotrienes have also been identified. Lipoxygenases are a group of enzymes, which oxidize polyunsaturated fatty acids possessing two cis double bonds separated by a methylene group to produce lipid hydroperoxides. Arachidonic acid is thus metabolized to a number of hydroperoxy- eicosatetraenoic acid derivatives (HPETEs). These enzymes differ in the position at which they peroxidize arachidonic acid and in their tissue specificity. For example, platelets possess only a 12-lipoxygenase while leukocytes possess both a 12- lipoxygenase and a 5-lipoxygenase. The HPETE derivatives are not stable, being rapidly converted to a number of metabolites. Leukotrienes are products of the 5- lipoxygenase pathway and are divided into two major classes: hydroxylated eicosatetraenoic acids (LTs) represented by LTB4 and peptidoleukotrienes (pLTs) such as LTC4, LTD4 and LTE4. 5-Lipoxygenase will produce leukotrienes from 5-HPETE as shown in Figure. LTA synthetase converts 5-HPETE to an unstable epoxide termed LTA that may be converted by the enzyme LTA hydrolase to the leukotriene LTB4 or by glutathione-S-transferase to LTC4. Other leukotrienes (e.g. LTD4, LTE4, LTF4) can then be formed from LTC4 and then reconjugation with glutamic acid, respectively. One mediator of inflammation, known as SRS-A (slow-reacting substance of anaphylaxis), is primarily a mixture of two leukotrienes, LTC4 and LTD4. The physiologic roles of the various leukotrienes are becoming better understood. LTB4 is a potent chemotactic agent for polymorphonuclear leukocytes and causes the accumulation of leukocytes at inflammation sites and leads to the development of symptoms - characteristic of inflammatory disorders. LTC4 and LTD4 are potent hypotensives and bronchoconstrictors. Because of the role played by LTs and pLTs in inflammatory conditions and asthma, it is not surprising that intensive research is being conducted in the area of inhibitors of leukotriene biosynthesis.

Biosynthesis of leukotrienes.

Non steroidal anti-inflammatory drugs (NSAIDs)

Inflammation is a complex process, which is developed with participation of inflammatory mediators, such as prostaglandins, leucotrienes, FAT and others. They derived from membrane phospholipids. Under the action of Phospholipase A2 is formed arachidonic acid, which can metabolized by 2 ways with participation cycloxygenase and lipoxigenase. There are two overall reactions catalyzed by cyclooxygenase. The first reaction is a cyclooxygenase reaction that requires two molecules of molecular oxygen. The end product of this reaction is the formation of Prostaglandin-G2. The second reaction is a peroxidase reaction that generates the final product Prostaglandin-H2.

COX is a dimeric enzyme. COX 1 & 2 enzymes are dimers (i.e. two subunits) and are membrane associated. All NSAID are COX inhibitors. The current COX-2 Hypothesis generally accepted by the pharmaceutical community is that COX-2 specific inhibitors will reduce pain, fever, and inflammation without causing gastrointestinal or renal injury. There are two central tenets to this hypothesis: 1) The prostaglandins that mediate inflammation, fever and pain are produced solely via COX-2. 2) The prostaglandins that are important in gastrointestinal and renal function are produced solely via COX-1. The toxicity of non-steroidal anti-inflammatory drugs (NSAIDS) in the GI and renal systems would therefore be due to a lack of selectivity of those drugs with respect to inhibition of COX-1 and COX-2. Thus, if COX-2 selective inhibitors can be made that will spare COX-1, then it should be possible to design a drug without the adverse side- effects. To do this, it is important to understand the biochemical and structural differences between COX-1 and COX-2:  The COX-1 gene product is 599 amino acids long,  The COX-2 gene product is 604 amino acids long,  COX-1 is 17 amino acids longer in the N-terminal,  The last 4-resides in the C-terminus for both COX-1 and COX-2 are identical,  COX-2 is 18 residues longer in the C-terminal, which allows it to also associate to the nuclear membrane,  COX-I has isoleucines (Ile) at positions 434 and 523 in the active site,  COX-2 has valines (Val) at positions 434 and 523 in the active site,

 Difference of a single methyl group between the valine and isoleucine amino acid gives rise to COX-1 having a smaller substrate binding pocket, than COX-2. A portion of this increased volume in the COX-2 binding site results in a side-pocket. All the NSAIDs are COX inhibitors. Only Aspirin (acetylsalicylic acid) covalently and irreversibly modifies both COX-1 and COX-2 by acetylating Serine-530 in the active site. Other NSAIDs inhibit COX reversibly.

Analgesic, antipyretic, and NSAIDs is a compounds of several chemical classes. The main groups of them are: 1. Salicylic acid derivatives (sodium salicylate, magnesium salicylate, phenylsalicylate, salicylamide, aspirin, salsalate, diflunisal), 2. Para-aminophenol derivatives (Acetaminophen), 3. Arylpropionic acids (Ibuprofen, naproxen, flubiprofen, ketoprofen, fenoprofen), 4. Indole and indende acetic acids (Indomethacin, sulindac), 5. Heteraryl acetic acids (diclofenac), 6. Anthranilic acids (fenamates, mefenamic acid), 7. Tricycles or Coxibs.

Salicylates 30

The use of salicylates dates back to the 19th century. Since then, numerous derivatives of salicylic acid have been synthesized and evaluated pharmacologically, yet only a relatively few derivatives have achieved therapeutic utility.

The derivatives of salicylic acid are of three types:

salicylates modified at the carboxyl group (type I):

Salicylic acid itself appears local irritating activity and high toxicity and can not be used internally. That’s why it was synthesized ethers or salts of salicylic acid. Most hydrolysis of these derivatives takes place in the intestine. Based on this Nencki introduced salol and so presented to the science of therapy the "salol principle." In salol, two toxic substances (phenol and salicylic acid) were combined into an ester that taken internally slowly hydrolyzes in the intestine to give the antiseptic action of its components. This type of ester is referred to as a full salol or true salol, when both components of the ester are active compounds. The salol principle can be applied to esters in which only the alcohol or the acid is the toxic, active or corrosive portion; this type is called a partial salol. Example of partial saloIs that contain an active acid is methyl salicylate.

salicylates modified at the hydroxyl group (type II):

salicylates containing nuclear substitutions (type III):

Most of these compounds are absorbed unchanged into the bloodstream. The most effective was acetylsalicylic acid. In addition to antipyretic, analgesic and anti- inflammatory properties, salicylates possess other actions that have been proven to be therapeutically beneficial: uricosuric activity, antithrombotic activity, NSAIDs might be protective against colon cancer. Thus the therapeutic utility of aspirin continues to increase. Unfortunately, a number of side effects are associated with the use of salicylates, most notable GI disturbances such as dyspepsia, gastro duodenal bleeding, gastric ulcerations and gastritis, bleeding, kidney dysfunction.

Salicylates structure-activity relationships (SAR)

1. The active moiety appears to be the salicylate anion, because benzoic acid itself has only weak activity. 2. The carboxylate anion is required for anti-inflammatory action. 3. Side effects of aspirin, particularly the GI effects, appear to be associated with the carboxylic acid functional group. 4. Reducing acidity of this group (converting to an amide - salicylamide) maintains the analgesic actions of salicylic acid derivatives, but eliminates the anti-inflammatory properties. 5. Substitution on either the carboxyl or phenolic hydroxyl groups may affect potency and toxicity. 6. Placing the phenolic hydroxyl group meta- or para- to the carboxyl group abolishes activity.

7. Substitution of halogen atoms on the aromatic ring enhances potency and toxicity. 8. Substitution of aromatic rings at the 5-position of salicylic acid increases anti- inflammatory activity (e.g., flufenisal, which has twice the potency of aspirin, and a lower incidence of gastric irritation), however, the agent diflunisal is three times more potent than aspirin, and not need to be given as often, reducing the incidence of gastric side effects. Absorption and Metabolism of salicylates. Since salicylates are weak acids absorption generally takes place primarily from the small intestine and to a lesser extent from the stomach by the process of passive diffusion of unionized molecules across the epithelial membranes of the GI tract. Thus, gastric pH is an important factor in the rate of absorption of salicylates. Any factor that increases gastric pH, e.g., buffering agents, will slow the rate of absorption since more of the salicylate will be in the ionized form. Topical preparations of salicylic acid are effective in that the rate of salicylate absorption from the skin is rapid. The major metabolic routes of esters and salts of salicylic acid are their conversion to salicylic acid which may be excreted in the urine as the free acid (10%). Salicylic acid is then metabolized primarily to two major inactive metabolites: salicyluric acid arises from conjugation of salicylic acid with glycine, and gentisic acid arises from p- hydroxylation mediated by cytochrome P450. These two metabolites are then excreted in the urine.

The Para-amino Phenol Derivatives 33

Application of aniline derivatives was implemented in medicine, when antipyretic and analgesic effect of aniline and acetanilide /antifebrin/ was found out by Chan and Hip in 1886.

But these compounds possess considerably high toxicity, which is caused by met hemoglobin formation in the organism. Having a goal to get a compound with low toxicity and longer and stronger analgesic activity, Phenacetine was synthesized, which wasn’t deprived toxicity. But a very important fact was cleared up: both acetanilide and phenacetine being metabolized in the organism cause N-acetyl-p-aminophenol /paracetamol/, which indicate that analgesic and antipyretic effect is due to this compound.

Study biological activity of the latter confirmed this hypothesis and although that it was synthesized 50 years ago it began to be used in medicine and now it is classified in widely used preparations.

Study of aniline derivative pharmacological activity has shown that aniline derivative chemical structure and pharmacological activity have the following connection:

1. Generally almost all the derivatives of aniline possess analgesic and antipyretic activity, but unlike non-narcotic analgesics, anti-inflammatory activity is very weak or completely absent. 2. Amino group one of the hydrogen’s substitution with the group of decreased basic properties leads to toxicity decrease related to aniline, but analgesic and antipyretic activities decrease either. For example, Acetanilide in analgesic doses is less toxic; it is toxic only in high doses. 3. Formanilide is easily hydrolyzed and possessing irritating property.

Formanilide 4. The solubility of higher derivatives of Acetanilide, consequently their activity and toxicity are lower either. For example, benzanilid is completely deprived of analgesic and antipyretic activity; salicylanilide is used as a fungicide agent.

Benzanilide Salicylanilide 5. Substitution of 2nd hydrogen atom with alkyl radical, increases toxicity, for example Exalgin is very toxic substance.

6. Hydroxylated derivatives of aniline, which are known as aminophenols /o-, m-, p-/ are less toxic. P-derivative of these is much more interesting: because it represents metabolism product of aniline, is less toxic and has high analgesic and antipyretic activity, but it is toxic as a medicinal agent and because of this it undergoes different changes. Corresponding anisidine and phenetidine have been obtained by phenolic 35 group methylation and ethylation, but it was revealed, that in case of free amino group is impossible to prevent met hemoglobin formation.

Anisidine Phenetidine 7. The most successful change of p-aminophenol is the amino group acetylation by N-acetyl para-aminophenol (acetaminophen, paracetamol) formation, ethyl ether of which is known as Phenacetine.

Acetaminophen Phenacetin Mentioned above phenacetin and paracetamol are used in medicine as painkillers and antipyretic agents, but due to its nefrotoxic effect phenacetin is currently excluded from the application. There are a number of acetaminophen analogues, which have been synthesized, but none are superior to acetaminophen itself. The acetaminophen analogue phenetsal (p- acetaminophenyl salicylate, Salophen) is a true salol, since it is metabolized to acetaminophen and salicylic acid.

Metabolism and Toxicity. Both acetanilide and phenacetin are metabolized to acetaminophen. Additionally, both undergo hydrolysis to yield aniline derivatives that produce directly, or through their conversion to hydroxylamine derivatives, which form met hemoglobinemia and hemolytic anemia. On the other hand, acetaminophen is metabolized primarily by conjugation reactions, the O-sulfate conjugate being the primary metabolite in children and the O-glucuronide being the primary metabolite in adults. A minor part of paracetamol undergoes by a cytochrome P450 (CYP450) mixed function oxidase system converted to a reactive toxic metabolite an N- 36 acetylimidoquinone, which has been suggested to produce the nephrotoxicity and hepatotoxicity. Normally this quinone is detoxified by conjugation with hepatic glutathione. However, in cases of large doses or overdoses of acetaminophen, hepatic stores of glutathione may be depleted by more than 70% allowing the reactive quinone to interact with nucleophilic functions, primarily -SH groups, on hepatic proteins resulting in the formation of covalent adducts, which produce hepatic necrosis, renal dysfunction. Various sulfhydryl-containing compounds were found to be useful as antidotes to acetaminophen overdoses. The most useful of these, N-acetylcysteine and methionin serve as a substitute for the depleted glutathione, by enhancing hepatic glutathione stores or by enhancing disposition by non toxic sulfate conjugation. N-acetylcysteine may also inhibit the formation of the toxic imidoquinone metabolite. In cases of overdoses, N-acetylcysteine is administered as a 5% solution. There is a preparation, where paracetamol is combined with the methionin (in mixture form), which is called pamaton.

General SAR for Arylalkanoic acids 1. All nonselective COX inhibitors possess a center of acidity. The relationship of this acid center to the carboxylic acid function of arachidonic acid is obvious.

2. The activity of ester and amide derivatives of carboxylic acids is generally attributed to the metabolic hydrolysis products. 38

3. The center of acidity is generally located one carbon atom adjacent to a flat surface represented by an aromatic or heteroaromatic ring. The distance between these centers is crucial since increasing this distance to two or three carbons generally diminishes activity. 4. Derivatives of aryl or heteroaryl acetic or propionic acids are most common. 5. Substitution of a methyl group on the carbon atom separating the acid center from the aromatic ring tends to increase anti-inflammatory activity. 6. The resulting α-methyl acetic acid, or 2-substituted propionic acid, analogs have been given the class name "profens". Groups larger than methyl decrease activity, but incorporation of this methyl group as part of an alicyclicring system does not drastically affect activity. Introduction of a methyl group creates a center of chirality. Anti- inflammatory activity in those cases where the enantiomers have been separated and evaluated, whether determined in vivo or in vitro by cyclooxygenase assays, is associated with the (S)-(+)-enantiomer. Interestingly, in those cases where the propionic acid is administered as a racemic mixture, in vivo conversion of the R-enantiomer to the biologically active S-enantiomer is observed to varying degrees. 7. A second area of lipophilicity that is generally non coplanar with the aromatic or heteroaromatic ring generally enhances activity. This lipophilic function may consist of an additional aromatic ring or alkyl groups either attached to or fused to the aromatic center. All above mentioned could be presented by follow scheme:

Arylpropionic acids

The general structure of this group is:

SAR

1. The maximum activity is obtained for the substitution at R1 is isobutyl group. The smaller substituents (methyl, ethyl) reduce the activity.

2. Maximal activity is found with R2 is methyl group. Smaller and larger groups diminish the activity (all profens).

3. Replacement of carboxyl group by an ester, alcoholic, amide, hydroxamic acid (NHOH) or tetrazole (CHN4) generally produce less active compounds. 4. The anti inflammatory activity resides in the S (+) isomer; R (-) isomer converted in the body to S (+) isomer. 5. Inserting the phenoxy group in the meta-position of aromatic ring arise the fenoprofen. Placing the phenoxy group in the ortho- or para-position of the arylpropionic acid ring markedly decreases activity. Replacement of the oxygen bridge between the two aromatic rings with a carbonyl group yields an analog (ketoprofen) which is also marketed.

6. Inserting of phenyl- group in the para position and fluor in the meta position of the atylpropionic acid leads to flurbiprofen, which was found to be more than 500 times more potent than aspirin.

7. 2-Naphthylpropionic acid derivatives possess anti-inflammatory activity. th Substitution in the 6 position with small lipophilic groups (CH30 being the most potent) led to maximum anti-inflammatory – Naproxen. (S) (+)-isomer is the more potent enantiomer. Naproxen is the only arylalkanoic acid NSAID marketed as optically active isomers

.Aryl- and Heteroarylacetic acid derivatives

1. Indole derivatives of acetic acid appear high potency.

2. N-benzoyl derivatives substituted in the para-position increase potency. 3. The most active is the chlorine-substituted derivative. 4. Methoxy substituted derivative is more active, than the unsubstituted indole ring. 5. Alkyl groups, especially methyl, at the α-position increase activity. Based on all above it was created indomethacin. 6. The resulting chirality introduced in the molecules is important. Anti-inflammatory activity is displayed only by the (S) (+)-enantiomer. The conformation of indomethacin appears to play a crucial role in its anti-inflammatory actions. The acetic acid side chain is flexible and can assume a large number of different conformations. The preferred conformation of the N-p-chlorobenzoyl group is one in which the chlorophenyl ring is oriented away from the 2-methyl group (or cis to the methoxyphenyl ring of the indole nucleus). This conformation may be represented as follows:

Indomethacin 7. The presence of indole ring nitrogen is not essential for the activity because the corresponding benzylidenylindene analogs (e.g., sulindac) are active.

8. The isosteric replacement of the indole ring with the indene ring system resulted in a derivative with therapeutically useful anti-inflammatory activity and less CNS and GI side effects, but which possessed poor water solubility and resulting crystalluria. 9. The replacement of the N-p-chlorobenzoyl substituent with a benzylidiene function resulted in active derivatives. 10. Replacement of the 5-methoxy group of the indene isostere with a fluorine atom enhances analgesic effects. 11. The decreased water solubility of the indene isostere was alleviated by replacing the chlorine atom of the phenyl substituent with a sulfinyl group. 12. Conjugation of arylacetic acid with the dichlorine aniline ring leads to very effective NSAID – diclofenac. The function of the two o-chlorogroups is to force the anilino-phenyl ring out of the plane of the phenylacetic acid portion, this twisting effect being important in the binding of NSAIDs to the active site of the cyclooxygenase enzyme.

13. N-Substitution of antranilic acid by dimethylphenyl radical leads to mefenamic acid.

Selective COX-2 inhibitors

As known COX-1 consist 599 amino acid and COX-2 604 amino acid. COX-1 has isoleucines (Ile) in the active site and COX-2 has valines in the active site. Difference of a single methyl group between the valine and isoleucine amino acid gives rise to COX-1 having a 25% smaller substrate binding pocket than COX-2. A portion of this increased volume in the COX-2 binding site results in a side-pocket that could possibly be filled by COX-2 selective NSAIDs.

In view of this it was synthesized a numerous tricyclic series of COX-2 inhibitors. They consist of a central ring with 1,2-biaryl substitutions.

In COX-I, the larger isoleucine residue near the active site restricts access by larger, relatively rigid side chain substituents, such as sulfamoyl or sulfonyl side chains usually seen in the selective COX-2 inhibitors. The sulfonyl moiety is the optimum for selectivity and can be either methylsulfonyl or a sulfonamide. The sulfonamide group inserts into the side pocket of COX-2 bordered by Val 523 and hydrogen bonds. For example, nimesulide is not tricyclic, but contain methylsulfonyl and appear the predominantly COX-2 selectivity.

Drugs acting on the CNS

Sedative-hypnotics

Sedative-hypnotics mainly are used to treat insomnia. They are also sedative medications and soporifics. The observed pharmacological effects of drugs in this class usually are dose related. Small doses cause sedation, larger doses cause hypnosis /sleep/ and still larger doses may bring about surgical /general/ anesthesia. The drugs currently used as hypnotics are effective, but there is ample need for newer, safer and more effective hypnotics. The ideal hypnotic should 1/ cause a sleep similar to physiological without altering sleep structure, 2/ have no potential for decreasing or arresting respirations /even at relatively high doses/, and 3/ produce no abuse, addiction, tolerance or dependence /abstinent syndrome/. Many physiological, autonomic and biochemical changes are take place during the sleep time. The centers in a brain are found, which participate in a sleep process, for instance hypothalamus, reticular formation etc. Sleep regulation mechanisms are not completely known, but there are a number of neurotransmitters /neuromodulators/ involved in sleep or wakefulness. There are: Catecholeamines are involved in wakefulness and rapid sleep. Initial experiments with reserpine suggested that a decrease in catecholamines involved in neurotransmission caused a decrease in rapid sleep. Compounds, which decrease or inhibit catecholamines synthesis, lead to sleep disorders. At the same time, α1-agonists decrease rapid sleep, α2-agonists are associated with sleep induction. Dopamine has a facilitative and active role in the sleep wakefulness cycle. Waking appears to be a state maintained by D2 activation, whereas decreased D2 activity appears to promote sleep. Serotonin. Initially, serotonin was thought to be a sleep-promoting neurotransmitter. Current studies indicate that conditions for sleep are now met when the serotoninergic system becomes inactive. The serotonin agonists for the 5-H1, 5-H2, 5-H3 receptors cause wakefulness and inhibit sleep, and serotonin receptors in the hypothalamus are involved in sleep regulation.

Histamine also has involvement in sleep. Histamine 1, 2, 3, receptors differ in molecule structure, distribution in the CNS, and physiology responses. The H-1 receptor agonists and the H-3 receptor antagonists increase wakefulness, whereas H-1 receptor antagonists /dimedrol/ and H-3 receptor agonists have the opposite effect. The H-2 receptors agonists and antagonists have not been shown to have any effect on wakefulness or sleep parameters.

Acetylcholine was the first neurotransmitter shown to have a role in wakefulness and initiation of rapid sleep. Conversely, atropine /cholinolytic/ decreases rapid sleep stage duration. Adenozin. The stimulation of the adenosine A1 receptors causes a hypnotic effect, thus, blocking of the central adenosine receptors with methylxanthines /caffeine/ is associated with wakefulness and a reduction in total sleep time. Melatonin, at times referred to as the “hormone of darkness”, normally is secreted during the night. The MT1 receptor appears to be primarily involved in initiating sleep, whereas the MT2 receptor appears to mediate melatonin’s effect in the eye and vascular effects. The importance of MT3 is currently unknown. Melatonin is most effective in young individuals and appears to be less effective in elderly individuals /possibly because of a decreased number of receptors/. Growth hormone, prolactin and CNS peptides also are involved in the sleep regulation. GABA /γ-amino butyric acid/ is the most important inhibitory transmitter of the CNS. In contrast to monoamines GABA quantities are high and being a γ-amino acid is not included in the proteins structure, in comparison with α-aminobutyric acid. In the body GABA is synthesized from the glutamate under glutamine acid decarboxylase enzyme, co-factor of which is pyridoxal phosphate. GABA molecules like other neuromediators accumulate in the vesicles of the presynaptic part. During a neurotransmission GABA undergoes exocytose into the synaptic hole and binds with GABA receptors. There are 3 types of GABA receptors A, B and C. There are postsynaptic and pre synaptic, central and peripheral GABA receptors. GABA-A is an ionic receptor. GABA-receptors have complex pentamere structure, which is obtained by α, β, γ, δ, ε, π, ρ and κ subunits different combination. These receptors types are differed not only by structure, but also location and sensitivity toward drugs and endogen ligands. GABA-A receptor of the CNS represents macromolecular transmembrane protein, which has a number of /until 16/ subunits 6α, 4β, 3γ, 1δ, 2ρ. It has extra cellular part consisted of 2 α, 2 β, and 1 γ, spirals and intra cellular part. Extra cellular part is for binding with ligands 2 molecules. GABA and its agonists, binding with the GABA-A receptors, lead to chlorine channels

45 opening in the result of which hyperpolarisation and synaptic response inhibition occur. GABA-B receptors are G protein-depended and don’t take part in sleep. GABA-A receptor has some binding parts such as benzodiazepine, barbiturate, alcoholic parts. Benzodiazepines, barbiturates, are in the allosteric interaction with GABA receptors complex. Those increase receptors sensitivity toward GABA showing CNS inhibiting /sedative, hypnotic/ activity. Based on mentioned above systems, which are included in a sleep process, hypnotic drugs can be classified into the following main groups: a/ GABA- receptors agonists Benzodiazepines Barbiturates Imidazopyridines and pyrazolopyrimidines derivatives b/ melatonin receptors agonists c/ anti-histamine drugs d/ antidepressants Benzodiazepins

O H N 1 N 8 9 2 9 1 3 8 2 7 4 6 5 3 N 7 4 6 5 N

3H-1,4-Benzodiazepine

1,2-dihydro-3H-1,4-benzodiazepine-2-on

Benzodiazepines are used as tranquilizer, hypnotic, anesthetic, anticonvulsant and spasmolytic agents. Action mechanism of this group of drugs is conditioned by binding with GABA-A receptors allosteric sites. When benzodiazepines bind to the GABA receptors α1-subunit they show sedative, amnesic and anticonvulsant activity, but anxiolytic and muscle relaxant activities binding with α2-subunit. Benzodiazepines bind with both GABA-receptors subunits. Sedative-hypnotic activity is due to binding with α1- subunit.

Structure activity relationship /SAR/

First time SAR was studied in the 5-phenyl-1,4-benzodiazepine-2-on derivatives by Sternbach. The following groups are necessary for the binding with receptors: Ring A. Generally 5-phenyl-1,4-benzodiazepin-2-on derivatives include an aromatic or hetero aromatic ring, which participate in π/π interaction with receptor’s aromatic amino acid residue. Substituting A ring by other heterocycle affinity toward receptors and activity decrease. Inserting radicals in this ring activity changes in different ways. Insertion of an electro acceptor group /halogen, nitro/ in the 7th position tranquilizer activity significantly increases. The stronger is radical’s electro acceptor properties the greater is activity. Insertion of radical in the 6, 8, or 9 positions tranquilizer activity sharply decreases. Ring B. 1. Insertion of phenyl group in the 5th position activity increases. 2. Electro acceptor group /carbonyl/ in the 2nd position is necessary for activity, because it binds with receptor’s histidine residue, which represents hydrogen donor. 3. Oxygen substitution with sulfur /in quizepam/ leads to more selective binding with benzodiazepine receptors subtypes. 4. Insertion of an alkyl group in the 3rd position activity decreases, but hydroxyl group increases. 3-hydroxi group containing derivatives comparatively are more active than their non-hydroxylated derivatives and excrete more rapidly from the body as they are polar and undergo glucuronidation easily. 3-hydroxy group esterification also is possible, which does not lead to activity decrease.

H3C O N

Cl N

Diazepam (Valium)

5. 1-N substitute must be small /methyl/. 6. 4,5 double bond saturation or its displacement to the 3,4 position leads to activity and affinity decrease. 7. If 1,2 positions saturate with ,,electron-rich,, /hydrogen acceptor/ ring, such as s- triazole or imidazole pharmacological activity will be increased, they will have high affinity toward the receptors.

N N

N N N

N N

s-Triazolo[4,3a][1,4]benzodiazepine Imidazo[1,5a][1,4]benzodiazepine

For instance, s-triazolo benzodiazepines: triazolam, alprazolam, estazolam and imidazo benzodiazepines: midazolam, are widely used as tranquilizer, sedative-hypnotic compounds. Ring C. 5-phenyl ring is not necessary for directly binding with benzodiazepine receptors, but extra aromatic ring can assist desirable hydrophobic or van der Waals interaction with the receptor and its interaction with ring A plane can be very important. Insertion of substitute in the 4th para-position into the 5-phenyl ring is undesirable for agonistic activity, but 2-orto or 2,6-diorto-positions substitution by electro acceptors increases activity. Physicochemical properties and pharmacokinetics These drugs are dissolved in lipids, due to which are completely absorbed in per os administration and rapidly reach to the brain, distribute almost in all organs. After absorption 70-99% is bound with blood plasma proteins. The onset of sedative-hypnotic 48 activity of intravenously administered benzodiazepines is rapid, with a range of 15-20 seconds to a few minutes.

O H3C H O N O H N N

OH Cl N Cl N Cl N

N-Desmethyldiazepam Diazepam (Valium) Oxazepam

The benzodiazepines are metabolized by liver microsomal enzymes and basically undergo N-dezalkylation and aliphatic hydroxylation, and then formed compounds undergo glucuronidation, in result of which hydrophilic compounds are formed and easily eliminated from the body via urine. For example, diazepam is rapidly absorbed by GIT and the highest concentration is noticed in the blood after 2 hours of administration, but half-life time is 20-50 hours. Diazepam forms N-dezmethyl diazepam by N-dezalkylation, which is pharmacologically active and it is metabolized more slowly than its parent compound. Double insertion leads to the N-dezmethyl diazepam accumulation, which can be found in blood even more than week later after administration. N-dezmethyl diazepam hydroxylation in the 3rd position leads to the active metabolite oxazepam formation. Nowadays oxazepam is used as separate short term tranquilizer preparation. It rapidly undergoes glucuronidation and eliminates via urine. In contrast to barbiturates benzodiazepines have some advantages: weaker dependence, abstinent syndrome is lighter. These drugs are safer; less activate metabolic enzymes, due to which tolerance is developed later, besides there is no contraindication to combine with other drugs. Barbiturates

Many somnolent, sedative, anticonvulsant agents are barbituric acid /pyrimidin 2,4,6- trion/ derivatives. Pyrimidin represents six membered heterocycle with 2 nitrogen atoms in 1,3 positions, one of the diazine isomers.

R R O 5 O 6 4 HN 1 2 3NH

Pyrimidine basic properties /despite 2 nitrogen atom presence/ is weaker in comparison with pyridine. Barbituric acid derivatives or cyclic ureids represent uric acid and malonic acid derivatives condensation product. In result of condensation closed cyclic system is formed with 2 nitrogen atoms /1,3 positions/, thus barbiturates are classified to the pyrimidine derivatives. The barbiturates are first generation sedative-hypnotics and beside sedative-hypnotic activity they exert a depressant effect on the skeletal muscle, smooth muscle and cardiac muscle. Depending on the compound, dose, and route of administration, the barbiturates can produce different degrees of CNS depression and have found use as sedatives, hypnotics, anticonvulsants or general anesthetics. Currently, the barbiturates get minimal use as sedatives and hypnotics because of higher toxicity. This is associated with their ability to cause greater CNS depression and their ability to induce many of the liver drug-metabolizing enzymes. In addition, the barbiturates cause tolerance and dependence. Mechanism of action At therapeutic doses, the barbiturates enhance the GABA-ergic inhibitory response in a mechanism similar to that of the benzodiazepines /i.e., by influencing conductance at the chloride channel/. At higher concentration, the barbiturates can potentiate the

GABAA-mediated chloride ion conductance and enhance both GABA and benzodiazepine binding. Therefore, the barbiturates and benzodiazepines display cross-tolerance. The barbiturate binding site is different from the benzodiazepines.

Structure activity relationship /SAR/

In 1951, Sandberg made his postulation that to possess good hypnotic activity; a barbituric acid must be a weak acid and must have a lipid/water partition coefficient between certain limits. Therefore, only the 5,5-disubstituted barbituric acid, or 1,5,5- trisubstituted barbituric acids possess acceptable hypnotic, anticonvulsant or anesthetic activity. All other substitution pattern, such as 5-monosubstituted barbituric acids, 1,3- disubstituted barbituric acids, or 1,3,5,5-tetrasubstituted barbituric acids, are inactive or produce convulsions. 50

2 types of tautomerization are typical to the barbituric acid: lactam-lactim and keto- enol (due to hydrogens of the imide group). The 5,5-disubstituted barbituric acid contains three lactam groups that can undergo only pH dependent lactam-lactim tautomerization because hydrogens at the 5th position are substituted by the radicals. Lactim form is responsible for the acidic properties of the barbituric acid derivatives. In presence of hydroxide ions they are dissociated like acids and form salts with metals.

The acidity of barbiturates in aqueous solution depends on the number of substituents attached to barbituric acid. The 5,5-disubstituted, and 1,5,5-trisubstituted barbituric acids are relatively weak acids and salts of these barbiturates are easily formed by treatment with bases.

R R R R R R O O O 5 O O O- 5 NaOH 6 5 + 6 4 4 NaOH 6 4 Na 1 3 HN 1 2 3NH pHa 7.1-8.1 HN 2 NH pHa 11.7-12.7 HN 1 2 3NH

- + O O Na O- Na+ Monolactim Dilactim

Barbituric acid derivatives used in medicine can be divided into 2 groups: barbiturates (lactam form) and barbiturates sodium salts (lactim form).

Acidic form Salt form

Barbital, phenobarbital, benzobarbital (benzonal) are barbiturates, and sodium- barbital, sodium-hexobarbital (hexenal), thiopental-sodium are sodium salts, which are different from each other by the R1, R2, R3 radicals.

5,5-Disubstitution. As the number of carbon atoms at the 5th carbon position increases, the lipophilic character of the substituted barbituric acids also increases. Hypnotic activity increases parallel with lipophilicity. In the 5th position optimal activity displays, when the number of carbon atoms in two substituents is 6-10. The number of carbon atoms further elongation leads to hypnotic activity decrease or inactivation, sometimes convulsion effect. Although lipophilic character determines the ability of compounds to cross the blood-brain barrier /BBB/, hydrophilic character also is important, because it determines solubility in biological fluids and ensures that the compound reaches the BBB. Substituents branching, unsaturation increase activity and decrease action duration. Insertion of polar group instead of alkyl group decreases

52 lipophilicity. Such modifications were of primary importance in the development of barbiturates with short duration of action.

Phenyl radical increases depressing effect, in result of which these preparations obtain anticonvulsant activity. Substitution on nitrogen. Substitution of one imide hydrogen by alkyl groups increases lipid solubility. The result is a quicker onset and a shorter duration of activity. As the size of the N-alkyl substituent increases /methyl, ethyl, propyl/ the lipid solubility increases and the hydrophilic character decreases. Attachment of alkyl substituents to both N1 and N3 renders the drug non acidic, making them inactive. Modification of oxygen. Replacement of C2 oxygen by sulfur increases lipophilicity. Because maximal thiobarbiturate brain levels are quickly reached, onset of activity is rapid. As a result, these drugs /i.e., thiopental/ are used as non inhalation /intravenous general anesthetics. Substitution of oxygen in the 2nd position in the Phenobarbital molecule by 2 hydrogen atoms increases anticonvulsant activity strengthening and decreases hypnotic activity. Metabolism. Barbiturates lose their activities through metabolic transformation. The metabolism of the barbiturates takes place primarily in the liver. After metabolism, the lipophilic character of barbiturates decreases and this is associated with a loss in activity. The major pathways include the following: a/ 2-thiobarbiturates desulfuration, b/ dezalkylation - lose of N-alkyl group, c/ 5th position substituent’s oxidation, d/ barbituric acid ring hydrolytic splitting. For example, Phenobarbital under CYTP450 is oxidized and in the first phase OH group joins with the aryl group. In the second phase glucuronides and sulfates are 53 formed. Barbiturates basically are excreted from the body through the kidneys in unchanged form or partially oxidized or partially conjugated in the side chain.

O O

NH 1 HO NH

O N O H O N O H Phenobarbital 2

R'O NH

O N O H R' glucuronide / sulfat

Nootropics

Nootropics are specific representatives of neuropsychostimulators /neuropsychotrops/, action of which is conditioned by their ability to improve study, memory processes and stimulate intellectual function in different disorders and pathological conditions and in healthy persons as well. The group name was formed from two Greece words noos-mind and tropes- direction. It was used by Giurgea at first time, after creation of Piracetam. Recent nootropics have unique mnemotrop influence; improve higher integrative functions of CNS, such as: intellect, concentration, long-term and short-term memory. They also increase memory possibility and information reproducibility, accelerate learning processes, improve spatial orientation, and increase resistance of brain tissue to physical and chemical undesirable influences. Nootropics also are used in human general biological action level decrease, which arises in different diseases and extreme conditions-ischemia, brain damages /from different origin/, intoxication, sleep disorders, tiredness, intellectual overload, pain syndrome, stress, perinatal influences etc. Moreover they must be non-toxic /greater therapeutic window/ and have fewer side effects, because they are used long term, besides they can be prescribed healthy people, for example in intellectual overload. Distinctly they must act by certain mechanisms, so it’s natural that there is no an ideal drug, which completely suits to this conditions, that’s why it is necessary to find out new drugs. Compounds relating to the nootropics have different chemical structure and action mechanism. As it was already mentioned nootropics prototype is Piracetam, which was pyrolidone derivative /cyclic GABA derivative/. After compounds of different structures 54 were followed, in which there were drugs acting on GABA, glutamatergic and other sistems and for them nootropic activity was not only the one activity, but also couldn’t be the main pharmacological activity, for example in case of GABA-ergic compounds /antihypoxic, sedative, anticonvulsant, tranquilizer/. Moreover, nootropic activity can be secondary conditioned by drug general pharmacological activity, for example can be formed in the result of brain general blood flow improvement, in brain ischemic states treating. From this point of view nootropics can conditionally be devided in §true¦ and §not true¦ groups. Classification Nootropics refer to the following classes: 1. Pyrolidone series /true/ nootropics /racemates/ pyracetam, oxyracetam, aniracetam, pramiracetam, etc. 2. Cholinergic agents /of different action- stimulating acetylcholine synthesis and release, anticholinesterase/ tacrin, physostigmine, galantamin, amiridin. 3. GABA-ergic drugs /GABA derivatives/ gammalon, pantogam, picamilon, phenibut, sodium oxybutirate. 4. Glutamatergic compounds- memantin, glycine, nooglutyl. 5. Neuropeptides and their analogues- semax, ebiratid, short dipeptides /pyracetam peptide analogues/ noopept. 6. Antioxidants. 7. Membrane protectors- mexidol. Ca channel antagonists, brain vasodilators and brain metabolism stimulators also can possess nootropic activity.

Structure activity relationship /SAR/ 1. Drugs which contain GABA moiety in their structure. Structure of GABA, one of the greatest inhibition mediators of CNS, plays the most relevant role in nootropics creation. Furthermore it is clear that GABA activates energy processes in brain, improve blood flow and increase glucose utilization by brain. Thereby it has nootropic and neuroprotective activity.

GABA

GABA has been used as nootropic agent, under the name ,,Aminalon,, according to his activity. Unfortunately it has not passed the BBB /as any amino acid it is hydrophilic

55 and it is in ionized zwitter ion form/ and has had fewer efficacies. The structure of GABA has been changed for developing his pharmacokinetics, lead to several new drugs: 1. Replacement of amino group to hydroxyl lead to creation of sodium oxybutirate, which has some nootropic efficacy, GABA’s activity alike, but unlike it he passes BBB easily. Besides this, it possesses hypnotic, myorelaxant and antihypnotic effect.

Sodium oxybutirate 2. In case of substitution by phenyl group in β-position we have Fenibut, which acts as nootropic in some cases, in addition to this it is tranquilizer.

Fenibut 3. Pantogam include GABA in his structure.

Pantogam 4. Picamilon is a yield combination of two well known drugs: GABA and nicotinic acid. The result of this combination is these drug’s pharmacological activity summary, such as spasmolytic and hypolipidemic activity of nicotinic acid with noopropic activity of GABA, so it makes Picamilon more efficient in treatment of brain atherosclerosis. It passes BBB easily too.

Picamilon We can see that these drugs are not completely selective nootropic agents, they have some other activity connecting with CNS, as well, and they are used as nootropics only in some cases.

2. The next group includes drugs which don’t have GABA in their structure or their structure is completely different from GABA. They have nootropic activity in despite of this. For example pyridoxine derivate: pyriditol or acefen.

Acephen Pyriditol 3. Drugs which are equal to GABA’s chemical structure. Piracetam /the first nootropic agent/ was discovered by chance in USB pharmaceutical company in Belgium in 1972, among several drugs which was using for “sea disease”. In the beginning, the scientists thought that piracetam’s pyrrolidine cycle is hydrolysed forming GABA in the organism, caused by his structure relation to GABA /it is cycling derivate of GABA/. This drug has possessed selective nootropic activity, and his discovering leads to new class of pharmacological active agents.

CH CH 2 2 O O N CH2 C CH C NH HO 2 2 NH2 O

GABA Piracetam

On the other hand it has some disadvantages, piracetam effect is not so powerful, it can be used in not deep deviations, and must be used in high dosses /1.2 g per day/. That is why many derivates were created based on this structure.

Oxiracetam Nefiracetam Pramiracetam

Aniracetam Nebracetam Etiracetam

By their nootropic activity, these preparations surpass the Piracetam, it has allowed lowering a dose, however these preparations cannot be ideal nootropics and they have a number of side effects.

At studying these preparations structure it becomes clear that in most cases 2- oxypyrrolidine moiety remains, thus clearly that it is necessary to have 2-oxypyrrolidine ring and peptide bond for the nootropic activity. Further, it has been proved that contrary to the theory, the piracetam molecule does not turn into GABA in the body, GABA quantity does not increase in CNS neither directly nor indirect mechanisms under piracetam and does not have expressed effect on the GABA receptors. 58

Considering these, in 1985 Russian scientists have assumed, that piracetam is a ligand of unknown receptors responsible for memory processes, inducing those receptors it possesses specific mnemotropic effect. This assumption has been proved by creation of antagonist compound, based on piracetam structure, and causes animals amnesia: L-proline cetyl ester. On the base of obtained data piracetam three pharmacophore groups were defined: α, β, γ. α

α- pyrrolidine ring β- carbamide group γ- lactam carbonyl α pharmacophore - change of the cycle sizes leads to activity decrease or loss. γ pharmacophore - absence leads to agonist conversion into antagonist, however this group can be entered as N-acetyl moiety, it will lead to agonist effects renewal.

β pharmacophore - can be attached to the cycle’s nitrogen or inserted in the 5th position.

Afterwards, some well-known neuropeptides (vasopressin, ACTH, etc.) influence was proved on the brain cognitive functions and their role in the memory processes. These peptides usage as preparations is impossible, because they are very unstable and don’t penetrate through the BBB. Piracetam has structural similarity with these peptides terminal amino acids: proline and pyroglutaminic acid: -the structure based on pyrrolidine ring in all three cases (proline, pyroglutaminic acid, piracetam) -piracetam and pyroglutaminic acid have carbonyl group in the 2nd position.

All these studies have served as a motivation for the Russian scientists to create a new class of potent, nontoxic nootropic agents: dipeptides, first representative of which is noopept. Dipeptides design Dipeptides were designed based on proline and pyroglutaminic acid structures. Why dipeptides were selected?: because bioavailability is very high in this case; in contrast to other more longer peptides, they don’t degradate in the GIT, well penetrate via biological barriers, BBB /they have high affinity toward brain tissue/; besides, amino acids of the content have specific biological activity and finally, these compounds are comparatively safe and common for the body. Piracetam and pyroglutaminic acid derivatives structures have been compared by computer conformation analysis design programs. It was found out that these groups’ pharmacophores have similarity with each other in the energetically convenient conformations.

These dipeptides showed high activity /in 1000 times less dose, than piracetam/. Proline containing dipeptides have been synthesized: aminoglycine moiety was replaced in the 2nd position; lactame carbonyl was replaced by acyl group; this

60 compound’s cycling leads to cyclo-prolyl-glycine formation, which was found as an endogenous nootropic peptide in the body.

O O N O C N H CH2CONH2 NHCH2CONH2

Piracetam glycine residue replacement to the 2nd position

O O C C N N

C NHCH2CONH2 C NH R O O

lactam carbonyl replacement by acyl group cyclo-prolyl-glycine

Based on this fact those derivatives have been synthesized, which will be metabolized by cyclo-prolyl-glycine formation in the body. Dipeptides general structure is shown as follows: Common structure of dipeptides

Noopept is the first representative of these compounds.

N-phenacetyl-L-prolyl glycine ethyl ester

It has high nootropic activity /approximately 2000 times more active, than piracetam/ and is metabolized in the body forming only endogenous compounds /toxicity absence/: cyclo-prolyl-glycine and phenyl acetic acid. Its specific effect is probably due to not only compound’s activity, but also the active metabolite. At therapeutic doses causes neither

61 stimulation, nor sedative effect. This preparation has already passed clinical trials and is registered under the name ,,noopept,, in Russia, but in US it was purchased by ,,Saegis,, company for further research.

Antipsychotics

Antipsychotics, known also as neuroleptics, and formerly called ataractics and major tranquilizers, not only produce calmness in severely disturbed psychiatric patients but also relieve them of the symptoms of their disease. However, contrary to the effect caused by hypnotics and sedatives, they do not cloud consciousness or depress vital centers. They are used in the treatment of patients with psychotic disorganization of thought and behavior, and in relief of severe emotional tension, especially schizophrenia. Over dosage is very seldom fatal in adults, and they do not induce psychological and/or physical dependence. Most antipsychotic agents also have sedative, tranquilizer, antiemetic, antihistaminic, cholinolytic and hypothermal activity. Because neuroleptic drugs potentiate the action of other central nervous system depressants, extreme care should be taken if a need arises to use them concurrently with alcohol, hypnotics such as barbiturates, narcotic analgetics, or general anesthetics. The most dangerous side effects of this group are extra pyramidal disorders. History. The search of ways to alter mood and behavior is as old as humans themselves. Alcohol and opium were probably the first drugs used for that purpose. Unfortunately, they do not help psychotic patients. Psychoanalysis, by its turn, developed at the beginning of the twentieth century, is not adequate for mass treatment. For this reason, until recently the only way to deal with psychotic patients was to isolate and restrain them physically.

The insulin-coma and electroshock therapy, developed some 60 years ago, was the first effective treatment of psychotic disorders and is still used nowadays. This discovery was followed 20 years later by the introduction of chlorpromazine and reserpine, the first antipsychotic agents. More than 5000 different phenothiazines have been synthesized up to date, some of which are used as antipsychotic and others as antihistaminic or as antipyretic agents.

Action Mechanism In the past several physicochemical mechanisms of action have been proposed for neuroleptic agents. Now it is believed that most antipsychotic agents act as antagonists at dopamine receptor, that is, by blocking dopamine from binding to its receptor sites.

Antipsychotic agents block both pre- and mainly postsynaptic dopamine receptors /D2/.

According to Janssen, all potent neuroleptics present two structural features in common, which he considers essential for high antipsychotic activity: 1. A steric chain of three carbon atoms linking the basic ring nitrogen with a carbon, nitrogen, or oxygen atom, this atom being a part of one of the following moieties: benzyl group, 2- phenothiazine or a thioxathene- tricyclic system, phenoxypropyl side chain or cyclohexane ring;

H2 H2 H2 N C C C Y

Y= C, N, O

2. A six – member basic heterocyclic ring, such as piperazine or piperidine, substituted in positions 1 and 4; the best substituents in position 4 are phenyl, aniline, methyl, or hydroxyethyl moieties. Notwithstanding their diversity of chemical structure, according to Janssen, all potent neuroleptics, because they present these two chemical features in common, act at the molecular level by the same mechanism. These two structural features give to a molecule similarity to a molecule dopamine. In complex formation with dopamine

64 receptors, at least two conformations of neuroleptics may be involved: (a) one, based on the solid-state conformation of chlorpromazine /cis/, overlaps almost exactly with an extended form of dopamine: and (b) a second, an “ S- shaped” conformation of the four-atom sequence that links an aromatic ring to the tertiary basic nitrogen atom in most neuroleptics.

The distance between nitrogen and aromatic ring is very important, which is only in the mentioned conformations corresponds to the distance between dopamine nitrogen and aromatic ring and provides receptor complete binding.

Classification According to the chemical structure antipsychotics can be divided into the following classes:  phenothiazine derivatives;  thioxanthene derivatives;  butyrophenone derivatives;  diphenylbutylamine derivatives;  substituted benzamindes;  indole derivatives,  arylpiperazine derivatives;  and miscellaneous agents.

Phenothiazine derivatives The general structure of all phenothiazine derivatives is shown in figure:

N R2

These compounds chemically represent lipophilic tricycle system, which is connected with hydrophilic aliphatic amino alkyl group by nitrogen atom of central ring. It is necessary to mention, that phenothiazine ring doesn’t have planar structure and corner between 2 phenyl rings is 141-159o. These compounds don’t solve in water and basically are used in hydrochlorides form.

Structure – activity relationship /SAR/

Synthesizing Phenothiazine various derivatives and studying their activity it becomes possible to study their structure-activity relationships. Changes in molecule can be divided into 3 basic groups: S 1. Substitution of the phenothiazine ring, 2. Changes in intermediate chain, N 3. Changes in aliphatic amino group. R2 R

CH2 CH CH2 N R Substitution of the phenothiazine ring R3 Inserting substituents in various positions of phenothiazine ring, it was cleared up that  The best position for the substitution is the 2nd position. Existence of substitute possessing hydrogen bond electron withdrawing ability is very important for its higher activity. It is due to the fact that hydrogen atom of aliphatic chain protonized amino group forms hydrogen bond with electron pair containing atom of the 2nd position. In a result the molecule obtains dopamine-like steric structure, which is necessary for receptor binding.

 As it was mentioned the activity increases at substituent insertion in the 2nd position, and the more are electron withdrawing properties the higher is compound nd activity. Thus CF3 subsitute containing compounds in the 2 position (Triflupromazine, Flurfenazine, triflurphenazine) are more active, than the corresponding chlorine derivatives. CF Cl 3 CF3

S NCH2CH2CH2N(CH3)2 S NCH2CH2CH2N(CH3)2 S N(CH2)3N NCH2CH2OH

Chlorpromazine Triflupromazine Fluphenazine (Permitil)

 Inserting thio methyl group in the 2nd position of phenothiazine ring in Thioridazine and mesoridazine their activity also increases, but preparation obtains side effects typical to thiol group containing compounds, such as pigment retinopathy, dermatitis. O SCH3 SCH3

S NCH CH 2 2 S NCH2CH2 N N CH 3 CH3 Thioridazine Mesoridazine (Mellaril) (Serentil)  Substitution at the 3rd position can improve the activity over non substituted compounds, but not as significantly as substitution at the 2 position; because H bond is getting weaker.  Substitution at position 1 has a deleterious effect on antipsychotic activity, as does (to a lesser extent) substitution at the 4 position. The effect of the substituent at the 1 position might interfere with the side chain’s ability to bring the protonated amino

67 group into proximity with the 2nd substituent. And as the sulfur atom at position 5 is in a position analogous to the p-hydroxyl of dopamine, and it was also assigned a receptor- binding function, the substituent at position 4 might interfere with receptor binding by the sulfur atom, which provides dipole-dipole /DD/ interaction with receptor. Changes in intermediate chain  The three-atom chain between position 10 and the amino nitrogen is required. Shortening or lengthening the chain at this position drastically decreases activity. The three-atom chain length may be necessary to bring the protonated amino nitrogen into proximity with the 2-substituent. S

N CH 3 CH2CH N CH CH3 3 Prometazine (Diprazine)

 Thus, in case of 2 carbon molecule they obtain antihistaminic effect /prometazine/.  Branching with large groups (e.g., phenyl) decreases activity, as does branching with polar groups (they are steric strain, inhibit molecule necessary conformation).  Methyl branching on the β-position has a variable effect on activity (molecule gets stereoselectivity). What is more important, the antipsychotic potency of L- (the more active) and D-isomers differs greatly. Changes in aliphatic amino group  dimethylamino group size decrease (substitution of monomethylamino or diethyl) greatly diminishes its activity;  piperidin and piperazin group insertion increases the effect (perphenazine);

 N10-acyl derivatives (ethmozine, ethacyzin, nonachlazine) in contrast to N10-alkyl derivatives show antiarrhythmic activity. S S

N NHCOOC2H5 N NHCOOC H C2H5 2 5

CCH2CH2 N C H CCH2CH2 N O 2 5 O Aethacizine O Aethmozine

 Derivatives which contain OH group in aliphatic amino group, converting into esters cause prodrugs, in the result of which duration of action is prolonged. For example Flurfenazine enantate and decanoate are flurfenazine prodrugs. The longer is

68 ester chain, the longer is action duration. Flurfenazine enantate is used once for 1-2 weeks, but decanoate - once for 2-3 weeks.

Thioxanthene derivatives Thioxanthene derivatives are obtained by isoster substitution of nitrogen atom in phenothiazene ring with double bond carbon atom. As phenothiazene ring thioxanthene ring doesn’t have planar structure, but corner between phenyl rings is sharper, 142- 150°. Compounds of this group possess activity similar to phenothiazene derivatives and their side-effects. As far as thioxanthene derivatives contain double bond, so they can be in cis and trans isomers forms, thus cis isomer is active, which corresponds dophamine active configuration.

All the mentioned changes in thioxanthene derivatives lead to similar biological activity change in thioxanthene derivatives. For example Flupenthixol decanoate is a prodrug, action of which is 1-2 weeks.

Antipsychotics belonging to other chemical groups Synthesizing more simple derivatives of compounds of three-cyclic structure the drugs with neuroleptic activity belonging to other chemical groups were obtained, in which SAR was studied. It was cleared up, that 1. All the necessary groups and dopamine-like structure also has butyrophenone derivatives, side effects of which in comparison with phenothiazine derivatives are expressed weaker. Preparations of this group have high affinity to brain dopamine D2 as well as to the serotonin 5-HT receptors. These compounds are selective inhibitors of dopamine D2 receptors.

2. For Butyrophenone derivatives activity it is necessary that tertiary amino group to be connected with aromatic ring by 4 carbon atom containing chain, which gives S- shape structure creation. Elongation or shortening of that chain lead to activity decrease. 3. Butyrophenone derivatives molecule should have such conformation that distance between F-substituted aromatic ring and nitrogen atom be 7.3 A, which is next to dopamine structure.

Haloperidol Dopamine 4. Substitution of keto group by thioketon, phenoxy group, or reduction also leads to activity decrease. 5. Tertiary amino group substituents change gives chance to obtain other derivatives possessing antipsychotic activity. For example, insertion of benzimidazole ring leads to droperidol creation, which has short-time duration and is used as a

F N N NH O sedative at anesthesia. Droperidol 6. Diphenylbutylamine derivatives were obtained by haloperidol molecule partial doubling and isosteric substitution, which have long-time action due to molecule lipophilicity increase and are used once a week per os in chronic schizophrenia. F

F R N Y R General structure of diphenylbutylamine derivatives 7. As far as piperazine ring was necessary for high activity (It was observed in Phenothiazine, Thioxanthene, Butyrophenone) so, that fact was based on creation of aryl piperazine group, which possess high antipsychotic activity. The representative of these compounds is Buspirone, which also has expressed tranquilizing activity. N N N O N N Buspirone hydrochloride O 8. The precondition for indole derivatives and isosters creation was the suggestion that serotonin plays an important role in schizophrenia mechanism development. Among the structural analogues of indole a wide range of preparations possessing neuroleptic activity were created and the important representative of which are molindone and sertindole. O

N O N H Molindone

Molindone has weaker affinity toward D2 receptors, than haloperidol, but in contrast to it, molindone doesn’t have extrapyramidal effects. CNS stimulants

CNS stimulants stimulate cerebral (especially that of the cortex) functions, facilitate inter-neuronal transmission of impulses, which leads to psycho-motor activities improvement. Due to the CNS stimulants activity the brain function and memory integration increase. And vice versa, a decrease of appetite, fatigue and sleep need is observed. At high doses they show analeptic activity, that is, they increase pulmonary ventilation and accelerate the return of normal reflexes, after anesthesia, for example. Classification According to their chemical structure CNS stimulants may be divided into 3 groups.  Phenylalkylamine derivatives – amphetamine sulfate,

Amphetamine  Sydnonimine derivatives – mesocarb /sydnocarb/,

mesocarb  Methylxanthines – caffeine, theobromine, theophylline.

caffeine theobromine theophylline Phenylalkylamines Mechanism of action for phenylalkilamines is: • block of NT reuptake, • direct agonist effects, • MAO inhibition. The simplest unsubstituted phenylisopropylamine is amphetamine. Amphetamine possesses central stimulant, anorectic, and sympathomimetic activities. The SAR for phenylalkylamines is relatively well-defined as well as for the NE agonists of indirect action. The phenyl ring and the distance between the amine and the phenyl are fairly strict.

CH3

CH2 CH

NH2

1. Substitution of the aromatic ring  Incorporation of substituents into the aromatic ring reduces or abolishes central stimulant activity.  The sympathomimetic agents of 4-hydroxyamphetamine are lack of central stimulant action and is unlikely to penetrate the blood-brain barrier due to the presence of the polar hydroxyl group. CH3

OH CH2 CH

NH2  Substitution of the hydroxyl group into methyl ether enhances the molecule lipophilicity due to which the compound displays weak stimulating activity which is ten CH times less that of amphetamine. 3 H CO CH CH 3 2 NH2  Halogen atom insertion weakens sympathomimetic activity and enhances the affinity of 5HT receptors. 2. Substitution of the amino group  Primary amines are more potent as central stimulants than the secondary amines, and the secondary amines are more potent than the tertiary amines.  As the length of the amine substituent increases, the activity decreases, however most of those drugs possess anorexic properties. 3. Substitution in alkyl chain  methyl group presence is the most optimal in the α-position, removal of which results in the agents with decreased lipophilicity and activity, also metabolic stability.  Homologation of the α-methyl group, to for example α-ethyl or α-propyl group, results in decrease of central stimulant activity.  The presence of α-methyl group creates a chiral center /stereoisomers/; S (+) isomer is several times more potent than its R (-) enantiomer.  β-hydroxylation of amphetamine results in decreased central stimulant activity, this may be the result of the decreased ability to penetrate the blood-brain barrier.

Methylxanthines Тhe large group of CNS stimulants is methylxanthine derivatives. Methylxanthines occured in nature, are caffeine, theophylline, and theobromine. Caffeine, theophylline, and theobromine are alkaloids which are present in tea leaf, coffee, cacao and cola fruits. From the earliest times humans have been using aqueous extracts of parts of some plants, containing caffeine, theophylline (“divine leaf”), and theobromine (“divine food”) for their CNS stimulating effects. Caffeine was first isolated in 1820 by Range and its first synthesis was carried out in 1897 by Fischer and by

Traube in 1900-1904. Since then several derivatives of caffeine and theophylline have been used in therapy as psychomotor stimulants. Action Mechanism  The CNS-stimulating effects of the methylxanthines were once attributed to their cyclic nucleotide phosphodiesterase-inhibiting ability, increasing cAMP quantity in the brain, heart and other organs tissues.  It is explained by the fact that the CNS-stimulating action is related more to the ability of these compounds to antagonize adenosine at A1 and A2A receptors.  They also promote NE release and promote intracellular Ca+2 releases. Caffeine is a widely used CNS stimulant /has higher activity from methylxanthine derivatives/. Because of central vasoconstrictive effects, caffeine is used for treating migraine and tension headaches (“Caffetamine”): Theobromine has very weak effect on CNS activity (probably because of poor physical chemical properties for penetration into the CNS). Methylxanthines are also reported to have bronchodilatating and diuretic properties. Theophylline and its preparations are widely used in bronchial asthma therapy. See Table 1 for their relative potencies.

Table 1. Relative Pharmacological Potencies of the Xanthines

Xanthine CNS Respiratory diuretic Coronary Cardiac broncho stimulant stimulant dilator stimulant dilator

Caffeine 1 1 3 3 3 3

Theophylline 2 2 1 1 1 1

Theobromine 3 3 2 2 2 2

1- Most potent. Caffeine is used as an analgesic in a combination with other analgetic agents in complex drugs such as pentalgin, sedalgin, cafetine. Its overdose leads to the development of tachycardia, nausea, vomiting, and convulsion.

Antidepressants

Antidepressants are those agents used to restore mentally depressed patients to an improved mental status. They are useful especially in depression and in depressive symptoms and, to a certain extent, in the treatment of depressive phases of certain types of schizophrenia. They decrease the intensity of a patient’s symptoms, reduce his tendency to suicide, accelerate the rate of his improvement, and promote his mental well-being. Various theories have been advanced to explain the biochemical causes of affective disorders. Most of them describe a fundamental role to neurotransmitter, such as dopamine, norepinephrine, and serotonin. Thus, according to the biogenic amine hypothesis, affective disorders result from genetically deficiencies in the functional activity of these neurotransmitters. History Until the introduction of electroconvulsive therapy in the 1930s, no treatment for depression was available. Antidepressants discovery was by chance, in 1951 it was observed hydrazine derivatives: isoniazid and its derivative iproniazid (isoniazid is an effective antitubercular agent but is a very polar compound). To gain better penetration into the Mycobacterium tuberculosis organism, a more hydrophobic compound, isoniazid substituted with an isopropyl group on the basic nitrogen (iproniazid) was designed and synthesized), used as tuberculostatic agents, elevated the mood of patients. In 1952, Zeller and colleagues found that iproniazide was able to inhibit the enzyme monoamine oxidase (MAO), which is responsible for monoamines inactivation. After several other pertinent effects were demonstrated by iproniazid, in 1957, Kline and co-workers employed this drug which success for the treatment of depressed patients. This discovery prompted the synthesis and pharmacological and clinical testing of many other hydrazine and hydrazide derivatives, some of which are being used as antidepressants. Iproniazid continued to be used in therapy for several years, but eventually was withdrawn because of hepatotoxicity.

- O C-NH-NH2 O C-NH-NH-CH (CH3)

N N Izoniazid Iproniazid

Simultenously, but independently, tricyclic compounds were introduced. The first to show useful antidepressant activity was imipramine, synthesized in 1957 as part of a program aimed at potential antihistamines, sedatives, analgesics, and anti-parkinsonism drugs in amino alkyl derivatives of iminodibenzyl. This preparation effect doesn’t depend

75 on MAO inhibition; Imipramine and other preparations of this group inhibit monoamines neuronal reuptake.

CH2-CH2-CH2-N(CH3)2 Imipramine In the last decade dozens of new compounds with a wide variety of chemical structures and pharmacological profiles have been developed. Great attention was paid to the compounds, which selectively inhibit only some monoamines: serotonin reuptake. Some authorities refer to these new drugs as “second-generation” antidepressants. They are structurally different from the tricyclic derivatives and MAO inhibitors, considered “first-generation” antidepressants. Examples of second-generation antidepressants are: fluoxetine, fluvoxamine, maprotiline, mianserin etc. An inorganic salts, lithium carbonate found its way into therapy more recently. The first observations of its calming effects in animals were made in 1949. After several years of investigation, it was finally confirmed that daily use of this drug is effective in preventing broad swings in mood in patients with manic-depression /however, lithium and strontium salts are basically normotymic /mood stabilizer/ preparations/.

Classification Antidepressants classify on mechanism of the action and chemical structure. Antidepressants can have a wide variety of chemical structures, and subdivided into following group: 1. Isonicotinic acid hydrazide derivatives: nialamide 2. Tricyclic antidepressants: a/ dibenzazepine derivatives: 1. dimethylamino compounds: tertiary amines /imipramine, clomiprmine, azaphene/ and 2. monomethyamino compounds: secondary amines /desipramine/, b/ dibenzocycloheptadiene derivatives: amitriptiline, damilene /tertiary amine/, nortriptylline /secondary amine/, 3. Tetracyclic compounds: maprotiline, mianserine. 4. Various chemical structure derivatives, such as: fluoxetine, sertraline, thianeptine, paroxetine etc. According to the action mechanism: 1. MAO inhibitors: a/ non reversible 76

b/ reversible: non selective and selective 2. Neuronal uptake inhibitors: a/ non selective b/ selective 3. Atypical antidepressants. Structure-activity relationship MAO inhibitors SAR

MAO, as you know, plays the physiological role of oxidative deamination primary and secondary amines to aldehydes, ammonia and hydrogen peroxide:

R-CH2-NH2 + O2 + H2O R-CH2O + NH3 + H2O2

MAO is divided into 2 classes A and B, and differing is substrate specificities and in response to drugs. Thus neutransmitter amines are deaminated by type A MAO. MAO inhibitors are found in different classes of substances: aminopyrazines, hydrazides, hydrazines, indole alkylamines, oxazolidones and others. Hence it is not wise to extrapolate the structure-activity relationships of one group of MAOIs to another group. Most of MAOIs are hydrazine or hydrazide derivatives. Hydrazine moiety is highly reactive and may form a strong linkage with MAO, inhibiting this enzyme for up to 5 days: nialamide causes non reversible inhibition. As a group these drugs /especially first generation non reversible drugs/ are less effective and produce more serious adverse reactions than TCA. For these reason they are drugs of second choice. More emphasis has to be selective and reversible inhibitors. Patients receiving anti-MAO drugs must avoid the ingestion of cheese or other tyramine containing food, lacked “cheese reaction”, hypertensive crises, and also they must avoid several types of drugs: alcohol, anesthetics, antihistamines etc.

Monoamine reuptake inhibitors /MRI/ SAR

Originally, the MRIs were a group of closely related agents, the tricyclic antidepressants, but now there are quite diverse chemically. Almost all of the agents block neuronal reuptake on NE /adrenaline/ or 5-HT /serotonin/ or both. Reuptake inhibition by these agents is at the level of the respective monoamine transporter via competitive inhibition of binding of the monoamine to the substrate- binding compartment. Probably the same site on the protein is involved for inhibitor and monoamine, but this has not yet been proved.

HO CH2-CH2-NH2

N H Serotonine /5-HT/

HO CH-CH2-NH2 OH

HO Norepinephrine /NE/

Tricyclic antidepressants /TCAs/ SAR

In summary, there is a large, bulky group encompassing two aromatic rings, preferably held in a skewed arrangement by a third central ring, and a three or, sometimes, two-atom chain to an aliphatic amino group that is monomethyl- or dimethyl- substituted.

phenothiazine derivatives tricyclic antidepressants

Tricyclic compounds are chemically similar to phenothiazine antipsychotic agents, and as in the case of those drugs, their antidepressant activity is related to structure, depending essentially on the: 1. tricyclic nucleus 2. the lateral chain 3. the basic amino group’s nature There are however, some differences between tricyclic antidepressants /TCAs/ and phenothiazine /Ph/ and thioxanthene antipsychotics: 1. The central ring of the tricyclic system of TCA is usually constituted of seven or eight atoms, which confers to them a more angled or twisted conformation than is found in PhA.

2. The tryciclic system is more out of plane than Ph ring systems. Thus the angle between two phenyl rings is between 1150 to 1300 for TCA, and between 1300-1500 for PhA.

3. Although the side chain in TCA is usually composed of the three carbon atoms, as it is a requirement in potent antipsychotic, strong activity is also present in some compounds in which this chain has only two carbon atoms. 4. The amino group in antidepressants is generally secondary, not tertiary as in antipsychotics. The overall arrangement has features that approximate a fully extended trans conformation of the β-aryl amines /NE, 5-HT/. To relate these features to the mechanism of action, reuptake block, visualize that the basic arrangement is the same as that found in the β-aryl amines, plus an extra aryl bulky group that enhances affinity for the substrate-binding compartment of the transporter. The overall concept of a β-aryl amine-like with added structural bulk, usually an aryl group, appears to be applicable to many newer compounds: swwmonoamines reuptake selective inhibitors—selective serotonin reuptake inhibitors /SSRIs/, selective norepinephrine reuptake inhibitors /SNERIs/- that do not have a tricyclic grouping. The TCAs are structurally related to each other and, consequently posses related biological properties that can be summarized as characteristic of the group. The dimethylamino compounds /DMA/ tends to be sedative, whereas the monomethyl relatives /MMA/ tend to be stimulatory. The DMA tend toward higher 5-HT and non selective NE reuptake block ratios: in the MMA, the proportion of NE uptake block tends to be higher and in some cases is considered selective NE reuptake. The compounds have anticholinergic properties, usually higher in the dimethylamino compounds. When treatment is begun with a dimethyl compound, a significant accumulation of the monomethyl compound develops as N-demethylation proceeds. The TCAs are extremely lipophilic and, accordingly, very highly tissue bound outside the CNS. Tricycle compounds are the most widely used drugs for the treatment of depressed patients, being more effective and less dangerous than MAO inhibitors. However their non selective representatives: imipramine, amitriptilline have a number of

79 side effects and contraindications. Since they have anticholinergic and noradrenergic effects both central and peripheral side effects are often unpleasant and sometimes dangerous. TCA generally used as hydrochlorides, which occur as water-soluble, white or colorless crystalline powder. Metabolism In vivo TCA undergo metabolism, yielding demethylated, hydroxylated, and conjugated metabolites. For example, imipramine forms several different metabolites, including desipramine which accumulates in the body and is responsible for the observed pharmacological action. Then dezipramine and imipramine are hydroxylated, N-oxidized and conjugated with the glucuronic acid. By the same process amitriptyline is N-demethylated to nortryptilline, which is active metabolite. They undergo the same transformations as imipramine.

*HCl N N

CH2-CH2-CH2-N(CH3)2 CH2-CH2-CH2-NHCH3 Imipramine is Imipramine the Dezipramine lead compound of the TCAs. It is also close relative of the antipsychotic phenothiazines /replaces the 10-11 bridge with sulfur/, and the compound is the antipsychotic agent promazine. As is typical dimethylamino compounds, anticholinergic and sedative effects tend to be marked. Metabolic inactivation proceeds mainly by oxidative hydroxylation in the 2nd position, followed by conjugation with glucuronic acid of the conjugate. Oxidative hydroxylation is not as rapid or complete as that of the more nucleophilic ring phenothiazine antipsychotics: consequently, appreciable N-demethylation occurs, with a buildup of desiparmine. The demethylated metabolite is less anticholinergic, less sedative, and more stimulatory and is a SNERI. Consequently, a patient treated with imipramine has two compounds that contribute to activity. The activity of desipramine is terminated by 2- hydroxylation, followed by conjugation and excretion. A second demethylation can occur, which in turn is followed by 2-hydroxylation conjugation and excretion.

*HCl *HCl N Cl CH -CH2 -CH2 -N -(-CH3)2 N(CH3)2 Amitriptilline Clomipramine 80

Clomipramine is up to 50 times as potent as imipramine in some bioassays. This does not empty clinical superiority, but it might be informative about trycyclic and, possibly other reuptake inhibitors. The chlorine replacing the H substituent could increase potency by increasing distribution to the CNS, but it is unlikely, that this would give the potency magnitude seen. It might be conjectured that a H bond between the protonated amino group (as in vivo) and the unshared electrons of the chloro- substituent might stabilize a β-aryl amine-like shape and give more efficient competition for the transporter. Amitriptiline hydrochloride is one of the most anticholinergic and sedative of the TCAs. Because it lacks the ring electron—enriching nitrogen atom of imipramine, metabolic inactivation mainly proceeds not at the analogous 2nd position but at the benzylic acid 10 position. As is typical of the dimethyl compounds, N-demethylation occurs, and nortriptyline is produced, which has less anticholinergic, less sedative, and more stimulant action than amitriptyline. Maprotilline hydrochloride is sometimes described as a tetracyclic rather than a tricyclic antidepressant. The description is chemically accurate, but the compound, nonetheless, conforms to the overall TCA pharmacophore. It is a dibenzobicyclooctadiene and can be viewed as a TCA with an ethylene-bridged central ring.

Selective serotonin reuptake inhibitors /SSRIs/ SAR

Structurally, the SSRIs differ from the tricyclics, in that the tricyclic system has been taken apart in the center. This abolishes the center ring, and one ring is moved slightly forward from the tricyclic “all-in-row” arrangement. The net effect is that the β-aryl amine-like grouping is present, as in the tricyclics, and the compounds can compete for the substrate-binding site of the serotonin transporter protein /SERT/. As in the tricyclics, the extra aryl group can add extra affinity and give favorable competition with the substrate, serotonin.

Many of the dimethylamino tricyclics are, in fact, SSRIs. Since they are extensively N-demethylated in vivo to nor-compounds, which are usually SNERIs, however, the overall effect is not selective. Breaking up the tricyclic system breaks up an anticholinergic pharmacophoric group and gives compounds with diminished anticholinergic effects. Overall, this diminishes unpleasant CNS effects and increases cardiovascular safety. Instead, side effects related to serotonin predominate.

O OCH2 F3C

F NH

O NHCH3 Fluoxetine Paroxetine In fluoxetine, protonated in vivo, amino group can form H-bond to the ether oxygen electrons, which can generate the β-arylamino-like group, the other aryl serving as the characteristic “extra” aryl. The S isomer is much more selective for SERT than for NET. The major metabolite is the N-demethyl compound, which is as potent as the parent and more selective (SERT versus NET). To illustrate a difference between selectivity for a SERT and a NET, if the para-substituent is moved to the ortho position (and is less hydrophobic, typically), a NET is obtained.

NHCH H 3

*HCl

Cl H

Sertraline Cl

F3C O CH3

N Fluvoxamine O NH2

In the paroxetine structure, an amino group, protonated in vivo could H-bond with the

–CH2-O- unshared electrons, a β-aryl amino-like structure with an extra aryl group results. The compound is a very highly selective SERT. As expected, it is an effective antidepressant and anxiolytic.

Inspection of sertraline reveals the pharmacophore for SERT inhibition. The Cl substituents also predict tropism for 5-HT. The depicted stereochemistry is important for activity. The E isomer of fluvoxamine (shown) can fold after protonation to the β- arylamino-like grouping. Here the “extra” hydrophobic group is aliphatic.

Local anesthetics

Similar to many modern drugs, the initial leads for the design of clinically useful local anesthetics /LA/ were derived from natural sources. As early as 15th century, the anesthetic properties of coca leaves (Erythroxylon Coca) became known. The active principle of the coca leaf however was not discovered until 1860 by Niemann, who obtained a crystalline alkaloid from the leaves, to which he gave the name cocaine. Koller introduced cocaine into clinical practice in 1884 as a topical anesthetic for ophthalmological surgery. After cocaine releasing, discovering the structure and synthesis, became clear that it represents methyl ecgonine benzoate, which is easily hydrolyzed (especially during sterilization in high temperature), forming ecgonine, benzoic acid and methanol.

COOCH3

N CH3 O C

Cocaine had one big disadvantage: its toxicity was very high. Studies of the cocaine biological activity and its hydrolysis products have shown that local anesthetic activity is due to anesthesiophor group, which includes the following structural parts: 1. aromatic ring (with radicals), which gives lipophilic properties to the molecule, 2. aliphatic amino group, which is responsible for the molecule’s hydrophilic properties, 3. intermediate chain, which connects molecule’s lipophilic and hydrophilic components. Chemically it represents benzoic acid connected with different groups (Rs- different aliphatic radicals) by ester, amide or thio ether bonds (X=O, NH, S).

R''' R' Ar C X (C)n N R'' O R''''

Each of this components’ role on the anesthetic activity have been studied. It becomes clear, that the aromatic ester group is so important; scientists thought that all aromatic esters have an anesthetic activity. Therefore many derivatives of benzoic acid have been synthesized, majority of which possess high anesthetic activity and low toxicity. Anesthesine is an example of this group, which represents ethyl ester of para- amino benzoic acid /PABA/. O

H2N C O C2H5 Anesthesin

This compounds’ disadvantage is that practically used their soluble salts possess strong acidic reaction, in which result they are used in water insoluble bases form, which can’t be used for infiltration or transmitting anesthesia and can be used only for superficial anesthesia, in powders, ointments forms for ulcers, burns and mucous inflammation anesthesia. Having a goal to increase PABA alkyl ethers water solubility, tertiary amino group inserted in the molecule alcoholic part. This amino group insertion increases these ethers basic properties, giving chance to get salts, solutions of which don’t have acidic reaction, but it’s near to basic. Besides, tertiary amino group insertion in the esters molecule increases their anesthetic properties. PABA diethylaminoethanolic ester hydrochloride /designed according to this principle/ is well known in literature under novocaine or procaine names. Novocaine also has anesthesiophor group, but it isn’t free from side effects: it has anti-sulfanylamide effect, causes allergic reactions. Its local anesthetic effect is quite short and it can’t be used for superficial anesthesia. Due to these reason new local anesthetics synthesis continues and doesn’t stop.

O C2H5 .HCl H2N C O CH2 CH2N C2H5

Novocaine

It was very important to study interaction types between novocaine and its corresponding receptor for new local anesthetics synthesis. It was found, that there are different bonds between receptor centers and molecule different components /charge transfer` induced dipole, dipole-dipole interaction, hydrogen bonding, van der Waals interactions and hydrophobic interaction and ionic bond/.

SAR Changes in the aromatic ring. In all local anesthetics as in novocaine carbonyl group’s activity and binding force with the receptor depends on carbon atom partly positive charge, due to which conjugation is possible between double bond’s π-electronic cloud and aromatic ring π framework to delocalize toward carbonyl group carbon atom.

Insertion of an electron-donating substituent in the aromatic ring increases carbon atom partly positive charge, due to which binding with the receptor is stronger which leads to stronger local anesthetics effect.

Electro-acceptor substituent (for example nitro group) decreases or loses local anesthetic property. Insertion of a methylene group between the aromatic moiety and carbonyl function is undesirable.

Local anesthetic activity increases in case of amino group presence in the para- position of the aromatic ring /novocaine/. NH2 substitution by NHR or NR2: anesthetic activity increases when R extends till C3; C3-C6 activity remains and more than C6 sharply decreases. According to this dicaine was created, which represents para- buthylamino benzoic acid’s dimethylamino ethyl ester.

Dicaine

Changes in the intermediate chain. The distance between ester oxygen and amino group’s nitrogen is very important for the local anesthetic activity, which is detected by the carbon atoms number in the alcohol chain. The elongations and branching of this chain increases local anesthetic activity, also toxicity /local, general/ sharply increases. Local anesthetic activity increases also in cyclic group presence in this part, but this compounds are not used either, because they have strong irritative properties. Corresponding amides are obtained substituting ester oxygen by NH, the most of which show higher activity, than

86 isosteric esters, but their toxicity also is high. From these compounds sovcaine is used, but it requires great caution because of high toxicity.

C2H5

O NH CH 2 CH2 N C

C2H5

N OC4H9

Sovcaine

Novocaine’s corresponding isosteric amide, which is known under novocaineamide name has low expressed local anesthetic activity, but has very high anti-arrhythmic effect. Novocaine thioisoster - thiocaine has 3 times higher local anesthetic activity.

O O

C C2H5 C C2H5 NH CH 2 CH2 N S CH 2 CH2 N

C2H5 C2H5 H2N H2N

Novocainamide Thiocaine

Changes in the aliphatic amino group. Chain elongation increases local anesthetic effect and local irritative property. Branching decreases toxicity, and local anesthetic effect. Cyclic radicals were inserted; nitrogen in a cycle, etc. Tertiary amino group can be represented in heterocycle. Benzoic and para-amino benzoic esters were studied which contain heterocyclic radicals, such as pyrol, pyrolyn, piperidin, piperazine etc. Piperidine derivatives are the most interesting. Benzoate methylpiperidinpropanol- meticaine is one of the strongest local anesthetics now.

CH CH H C CH H2C CH 2 2

N N N CH R

CH CH H C CH H2C CH 2 2 Pyrol Pyrolin Poperidin

H2C CH2 CO O CH2 CH 2 CH2 N CH2

CH CH2

H3C

Meticaine

Amino acids amides are very interesting from which lidocaine has expressed local anesthetic effect, which is known as xylocaine.

Lidocaine Trimecaine

Action Mechanism Local anesthetics prevent nerve impulse formation and transmission. They block sodium channels, inhibit action potential formation and nervous impulse isn’t transmitted. Now it is known that there are specific receptors in the sodium channels, to which local anesthetics are bound.

Way of reaching to the receptor depends on their physico-chemical properties: molecule size, pK, solubility and binding by the chemical properties. Protonized molecules and quaternary ammonium compounds reach to their targets through the external hydrophilic way, which is possible only during channel activation. Lipophilic anesthetics are diffused across the nerve membrane in basic, uncharged state. They can reach to the receptor immediately: in their basic uncharged form- by hydrophobic way, also they can bind with medium protons and reach to the receptor after protonization - by hydrophilic way.

There is a hypothesis, according to which local anesthetics receptors represent acidic phospholipids, to which local anesthetics bind (especially with lecithin). Let’s study that binding for novocaine and receptor example; it is carried out: a/ via ionic –dipole interaction, where ionic group and polarized group take part in the bond formation. b/ via hydrophobic bond where lipophilic groups take part. Hydrogen bonds are also formed, the stronger is the bond, and the longer is the local anesthetic effect. It is known, that local anesthetics compete with Ca, which joining with the acidic phospholipids regulates sodium permeability through the nervous membrane. Local anesthetics forming mentioned above bounds displace Ca, causing cellular membrane structural and functional changes and impulse transmission is inhibited. A lot of local anesthetics can form a π-complex with thiamine, where the anesthetic appears as an electro donor and thiamin as an acceptor. As it is known, thiamin and its precursors (phosphate ethers) have fundamental importance in nervous excitability. Local anesthetics forming thiamine complex, block nervous transmission and develop local anesthesia.

Classification All local anesthetics can be divided into 2 groups: Ester derivatives. There are esters of the following acids: benzoic acid, para-amino benzoic acid, meta-amino benzoic acid or para-alkoxy benzoic acid. As esters drugs of this group easily undergo hydrolysis in vivo and in vitro losing their activity. More common classification is: a) benzoic acid derivatives, cocaine, isobucaine, pribecaine, propanocaine, etc. b) amino benzoic acid derivatives, betoxycaine, novocaine, tetracaine, benzocaine, etc. Amid derivatives. This group includes 3 subgroups:

a) basic amides - dibucaine, b) annelids – lidocaine, trimecaine, c) tertiary amides – oxetazine.

Lidocaine Trimecaine First 2 groups can be watched by the following way, when –COO- group is substituted by isosteric NHCO- group. In a result of this change stability increases and don’t undergo hydrolysis easily. The most used drugs are bupivacaine, octacaine, lidocaine, pentacaine, etc. Anesthesin, novocaine and dicaine are used as local anesthetics, but novocainamide as an anti-arrhythmic. Anesthesine is prescribed as 5-10% ointments, oil solutions, candles, and 0.25-0.3 pills and powders. Novocaine has a large application in infiltration and subdural anesthesia in the 0.25-0.5% water solution form, which is injected subcutaneous, i/m. Dicaine is stronger than novocaine, but nearly 10 fold is toxic. This is the reason that it is classified among the A list of drugs. It is used in ophthalmology and otorhinolaringology for superficial anesthesia in 0.5-2% solution form, as well as 0.3% solution for peridural anesthesia. As it was mentioned before, local anesthetics are esters of aromatic acids and amino alcohols. A lot of compounds have been studied later where amino alcohol residue was changed into amino acidic, and aromatic acidic residue was changed into alcoholic. In this case molecule intermediate part is ‘’turned’’, replacement around carbonyl oxygen and methylen group. This basic components change doesn’t effect on molecule carbonic skeleton. In both cases it contains complex ether group and tertiary amino group. Although, ester molecule components are radically differ from each other; in one case amino group carrier is alcohol, in the other case acid. These compounds are conditionally called compounds with ‘’reversed’’ complex ether group. It becomes clear that the local anesthetic effect remains by replacing of alcohol and acidic groups, but toxicity and cholinolytic activity decrease.

O R1 H2 C C N O (CH2)n R2 O R1 H2 C C N O (CH2)n R2

Complex ethers with ‘’reversed’’ ester group practically aren’t used, while their amides have found large application. Such change in the molecule leads to stability and they hardly undergo hydrolysis. There is no cross sensitivity between this group and benzoic acid derivatives. Lidocaine /xylocaine/ is widely used from this group. Metabolism. Generally, local anesthetics are divided into esters (novocaine) and non esters (Lidocaine). Esters are hydrolyzed under esterase’s, which are widely spread in the body. Thus these compounds can be metabolized in the blood, kidneys, liver forming p-amino benzoic acid and corresponding alcohol. If we use drugs with local anesthetics /LA/, which are also hydrolyzed by esterases (anticholinesterase agents, or atropine like drugs) there can be interactions because these drugs either will inhibit or will compete with LA for esterases. In result of which, LA action duration and toxicity will increase. Another incompatibility is between LA and sulfonamides. For example, PABA, formed after novocaine hydrolysis, competes with sulfonamides and inhibits sulfonamides’ activity. LAs of non ester type are metabolized under microsomal enzymes in the liver. 10% is excreted in unchanged by urine. It was cleared that lidocain side effects on the CNS is conditioned by monoethylglycine xylidine and glycine xylidine, which are formed when ethyl groups are separated from lidocaine molecule after penetrating BBB. Glycine xylidine has low anesthetic activity but high toxicity, because of slowly elimination from the body and accumulation can lead to intoxication. Lidocaine metabolic pathways

Decreasing this undesirable effects tocainide and tolycaine were synthesized which have high LA activity and have no side effects on the CNS. Tocainide instead of N-ethyl group has α-methyl group and due to which amino group degradation under amino- oxidases is prevented and has LA desirable properties. Tolycaine has o-carbomethoxy group, which is substituted instead of lidocaine o-methyl group. Carbomethoxy group is hydrolyzed easily in the blood and polar carboxyl group is formed, which can’t penetrate through BBB, thus will not have undesirable effects on the CNS, though it has N-ethyl group.

O C2H5 O NH2 H2 H2 NH C C N NH C C CH CH C2H5 3 H C COOCH H3C CH3 3 3

Tocainide Tolycaine

Tocainide and Tolycaine are used as antiarrhythmic agents.

Lidocaine is widely used in superficial, infiltration, transmission anesthesia /epidural, spinal/, in ointments, gels, aerosols, eye drops form. In i/v infusion form it is also used for epilepsies.

DRUG DESIGN CHARACTERISTICS FOR TREATMENT OF EXTERNAL /EXOGENOUS/ REASONS CAUSED DISEASES

The disease reasons can be either body’s different organ systems or biochemical processes disorders (endogenous reasons) or external (exogenous) factors.

External (exogenous) reasons are physical: radiation, heat, chemical: environmental toxins and biological: microbes (prions, viruses, bacteria, fungi, parasites), which cause different disease in the body. Of these categories of pathogens, microbe-induced infectious diseases are probably the most important.

Designing drugs for exogenous pathogens has some fundamental differences from the process of designing drugs to achieve the manipulation of endogenous processes. Exogenous pathogens represent targets for drug design that are non self, while endogenous targets are part of human body 93 and are called self. When a drug binds to a receptor in the human heart, the target is self; however, when a drug binds to bacteria within lung tissue, then the target is non self. The toxicity that the drug can inflict upon the surrounding tissues of the receptor microenvironment is quite different between self and non self. It is desirable for a drug binding to a bacterium to kill that bacterium; it is undesirable for a drug binding to heart tissue to kill cardiac cells. For antibacterial drugs selective toxicity is the key concept.

The types of intermolecular interactions exploited during drug design against non self exogenous targets may also be different. For example, it is acceptable for a drug to bind to a non self target by a covalent bond. Whereas drug–receptor interactions via covalent bonding are typically avoided for endogenous targets for reasons related to toxicity, covalent bonds are acceptable when targeting a non self receptor on an exogenous pathogen. This observation is well exemplified by the example of antibacterial agents, such as penicillin, that covalently link to the bacterial cell wall.

In developing drugs for the treatment of diseases caused by microbes, drug design strategies may differ widely from microbe to microbe. In terms of structural complexity, microbes exist on a structural spectrum (prions, viruses, bacteria, fungi, parasites), with prions being the least complex and parasites the most complex. Drug design for prion and viral diseases is the most challenging, since these microbes are structurally simple. A prion is merely a protein; viruses are composed principally of nucleic acids. Because of the structural overlap between prion proteins and viral nucleic acids and the corresponding macromolecules found in humans, it is difficult to design a drug specific for the microbe. At the other end of the spectrum, parasites have a structural complexity (organs, rudimentary nervous system) that begins to approach the sophistication of human cell lines. Because of this similarity, it may be difficult to identify a target that will enable selective killing of the parasite without causing concomitant harm to the host organism. The structurally intermediate bacteria have sufficient complexity to enable drug design without the complexity overlapping with that of the host biochemistry. Accordingly, antibacterial drug design has traditionally been more successful than drug design targeted against the other microbes. In designing drugs for exogenous pathogens, it is sometimes possible to minimize or even neglect pharmacokinetic design considerations. For instance, if a drug were being designed to treat intestinal parasites, it would be beneficial to ensure that the drug is restricted to the gastrointestinal tract and is never absorbed. The strategic use of charged or polar substituent groups to influence drug solubility and absorption can be manipulated under such circumstances to reduce the likelihood of drug absorption.

When designing drugs to contend with exogenous pathogens such as infectious microbes, it is immensely useful to understand the biochemistry of the microbe and how that biochemistry differs from human biochemistry. Such data are useful when designing a drug to kill a microbe without harming the human host. Understanding the genome of a microorganism enables insights concerning its biochemical operation and the identification of a potential drug gable target; comparing the microbe’s genome to that of a human enables an appreciation of whether that target is shared between the microbe and humans.

Antibacterial drugs

Bacteria have been killing humans for millennia. Many diseases (pneumonia, meningitis, gangrene) are caused by bacterial infections. One of the greatest triumphs of medicinal chemistry in the 20th century was the discovery of antibacterial drugs.

History

Since Luis Pastor /1822-1895/ in the end of 19th century had proved that some microbes can cause diseases in human and animals, Robert Koch had studied and described some pathogen microbes and suggested ,,Koch postulates,, people started frightening against microbes searching and creating antimicrobial drugs.

First synthetic selective antimicrobial agents were discovered earlier than antibiotics. Their creation was possible due to great scientist Paul Erlikh /1854-1915/. When he was studying different life tissues painting /1900/ he mentioned that there are dyes which paint only certain tissue and proposed to get such dyes, which dye only microbes killing them, but don’t act on other tissues /,,magic bullet conception,,/. These compounds will recognize microbe in the human organism and will damage. Years later P. Erlikh obtained such compound /number 606 from his synthesized/, which was less toxic for human and had antimicrobial effect. It was called salvarsan /lat. salvare- survive, arsenicum-arsen/. It had significant effect on treponema against syphilis.

It was not only new preparation obtaining but also chemotherapy birth. In 1906 syphilis microbe– treponema was released by Hoffman and Shaudin toward which salvarsan also were active and in 1908 Erlikh get Nobel Prize for these achievements.

But antimicrobial drugs real century started when one of the scientists G. Dogmak /1895-1964/ was studying a dye dark red /red streptocide/ which was created and patented under Prontosil name in ,,Farben Indastrie,, now ,,Bayer,,. Animals contaminated by staphylococcus aureus were not died, even if there are injected microbes 10 fold dosages. It is interesting that experiments on the human in the first time were carried out on scientist little daughter, who was contaminated by streptococcus and this antimicrobial preparation saved children life.

These preparations saved many human lives in world war years, but the other hand ,,sulfanilamide’s elixir,, /streptocide ethylene glycol preparation/ child’s medicinal form took over 100 children life in US /it was toxic/, which induce US congress FDA /Food and Drug Administration/ be engaged by drugs safety control.

Prontosil was inactive in vitro, but it was effective in life organisms. In 1935 Trufo and co-authors studying sulfonamides azo dyes discovered that animals’ urine which took these preparations is active in vitro and later becomes clear that activity provides their active-p-amino benzene sulfonic acid amide, discolored metabolite, which is formed in liver, by diazo-bridge reducing decomposition. This fact proved Fuler, who in 1937 released prontosil metabolite - sulfanyl amide /white streptocide/ from blood and urine. Thus sulfanyl amides - first synthetic per oral antimicrobial agents’ century started, and first time prodrugs conception was appeared. It was cleared that white streptocide was synthesized in 1908 by Helmo, and approximately 25 years was used as dyes precursor.

The sulfonamide antimicrobial drugs were the first effective chemotherapeutic agents that could be used systemically for the cure of bacterial infections in humans. Their introduction led to a sharp decline in the morbidity and mortality of infectious diseases. The rapid development of widespread resistance to the sulfonamides soon after their introduction and the increasing use of the broader- spectrum penicillins in the treatment of infectious disease diminished the usefulness of sulfonamides. Sulfonamides creation was the beginning of chemotherapy and due to these preparations first time appeared anti metabolites conception, which is widely used in drug rational design.

Antimicrobial century next great discovery belongs to Alexander Fleming /1881-1955/, who discovered penicillin, when worked in London St. Maria hospital in 1928, by chance he discovered that penicillium fungi inhibited microbes’ growth.

Scientist continued his researches in US with his co-authors /Chein and Florin/, where started penicillin production /Merck and Co company/. In the beginning penicillin was not achievable to all, because penicillin had short action duration and it should be injected frequently. In this period of time it was accepted method from penicillin using human urine release drug and usage.

In 1944 Dorothy Hojkin cleared penicillin structure by roentgen radiation analysis, which was based on antibacterial drugs most important compounds-β-lactams /penicillins, cephalosporines, carbapenems, oxacefamicines, monobactames and clavulonic acids/ creation.

Afterwards many scientist studied different microorganisms to discover new natural effective antibiotics classes. In this sphere Selman Vaxman had great achievement /1888-1973/ discovered aminoglicosides and other antibiotics. He used total screening method, in result of which streptomices griseus was created, from which Streptomicin and Neomicin B were obtained.

Thus 2 years later after 2nd world war Merk and Co pharmaceutical company was producing 2 antibiotics-penicillin and streptomycin, which were half of all seller drugs volume.

Antibiotics’,, golden century,, is in 1942-1962. In this period of time using screening method new classes were discovered, but in 70-90ies new generations of famous classes’ antibiotics were created. From the 90ies till now for new antibiotics discovery use not total screening methods but screening based on target, SAR studies, microbes genomes studies etc.

Antibiotics and antimicrobial drugs

Currently there is a wide variety of agents available for the treatment of bacterial infections. A broad spectrum agent works against many types of bacteria. A bacteriostatic agent does not kill bacteria but does inhibit their reproductive growth; a bactericidal agent actually kills bacteria. The term antibiotic is frequently used interchangeably for antibacterial. The word antibiotic, proposed by Waksman in 1942, refers to a substance that is able to inhibit the growth or even destroy microorganisms; the term is derived from Vuillemin’s concept of antibiosis (which literally means “against life”). The designation of antibiotic can thus be applied not only to antibacterial but also to other antimicrobials such as antifungal agents. In the strictest sense, antibiotics are antibacterial substances produced by various species of microorganisms (bacteria, fungi, and actinomycetes) that suppress the growth of other microorganisms. Common usage often extends the term antibiotics to include synthetic antimicrobial agents, such as sulfonamides and quinolones. Antibiotics differ 97 markedly in physical, chemical, and pharmacological properties, in antimicrobial spectra, and in mechanisms of action. As a conclusion: antibiotics are microbial metabolites or synthetic analogues inspired by them that in small doses inhibit the growth and survival of microorganisms without serious toxicity to the host.

Antibiotic are among the most frequently prescribed medications today. In many cases, the clinical utility of natural antibiotics has been enhanced through medicinal chemical manipulation of the original structure, leading to broaden antimicrobial spectrum, greater potency, lesser toxicity, more convenient administration and additional pharmacokinetic advantages.

Antibacterial drugs nomenclature

The names given to antimicrobials and antibiotics are as varied as their inventor’s taste: however, some helpful unifying conventions are followed. For example, the penicillins are derived from fungi and have names ending in the suffix –cillin, as in ampicillin. The cephalosporins likewise are fungal products, although their names mostly begin with the prefix –cef (or, sometimes, following the English practice, ceph-). The synthetic fluoroquinolones mostly end in the suffix –floxacin. Although helpful in many respects, this nomenclature does result in many related substances possessing quite similar names. This can make remembering them burden. Most of the remaining antibiotics are produced by fermentation of soil microorganisms belonging to various Streptomyces species. By convention, these have names ending in the suffix –mycin, as in streptomycin. Some prominent antibiotics are produced by fermentation of various soil microbes as Micromonospora sp.; these antibiotics have names ending in –micin, as in gentamicin.

Factors that influence on antibacterial drugs efficiency

Successful antimicrobial therapy of an infection ultimately depends on the concentration of antibiotic at the site of infection. This concentration must be sufficient to inhibit growth of the offending microorganism. The concentration of drug at the site of infection not only must inhibit the organism but also must remain below the level that is toxic to human cells. If this can be achieved, the microorganism is considered susceptible to the antibiotic. If an inhibitory or bactericidal concentration exceeds that which can be achieved safely in vivo, then the microorganism is considered resistant to that drug.

Bacterial resistance to antimicrobial agents

The resistance is the failure of microorganisms to be killed or inhibited by antimicrobial treatment. Resistance can either be intrinsic (exist before exposure to drug) or acquired (develop subsequent to exposure to drug).

The recent emergence of antibiotic resistance in bacterial pathogens, both nosocomially and in the community is a very serious development that threatens the end of the antibiotic era. Today, more than 70% of the bacteria associated with hospital-acquired infections in the United States are resistant to one or more of the drugs previously used to treat them. In some cases additional compensatory mutations can occur that restore the vigor of the resistant organisms. Resistance of this type usually is expressed toward other antibiotics with the same mode of action and, therefore, is a familial characteristic—most tetracyclines for example, show extensive cross-resistance with other agents in the tetracycline family.

For an antibiotic to be effective, it must reach its target in an active form, bind to the target, and interfere with its function. Accordingly, bacterial resistance to an antimicrobial agent is attributable to three general mechanisms:

 The drug does not reach its target (1)—failure of the drug to penetrate into or stay in the cell

 The drug is not active (2)—destruction of the drug by defensive enzymes

 The target is altered (3)—alteration in the cellular target of the enzymes

(1) The outer membrane of gram-negative bacteria is a permeable barrier that excludes large polar molecules from entering the cell. Small polar molecules, including many antibiotics, enter the cell through protein channels called porins. Absence of, mutation in, or loss of a favored porin channel can slow the rate of drug entry into a cell or prevent entry altogether, effectively reducing drug concentration at the target site. If the target is intracellular and the drug requires active transport across the cell membrane, a mutation or phenotypic change that shuts down this transport mechanism can confer resistance. Bacteria also have efflux pumps that can transport drugs out of the cell. Resistance to numerous drugs, including tetracycline, chloramphenicol, fluoroquinolones, macrolides, and -lactam antibiotics, is mediated by an efflux pump mechanism (Figure 42-1 depicts the multiple membrane and periplasm components that reduce the intracellular concentrations of lactam antibiotics and cause resistance.

(2) Drug inactivation is the second general mechanism of drug resistance. Bacterial resistance to aminoglycosides and to -lactam antibiotics usually is due to production of an aminoglycoside- modifying enzyme or -lactamase, respectively. A variation of this mechanism is failure of the bacterial cell to activate a prodrug. This is the basis of the most common type of resistance to isoniazid in M. tuberculosis

(3) The third general mechanism of drug resistance is target alteration. This may be due to mutation of the natural target (e.g., fluoroquinolone resistance), target modification (e.g., ribosomal protection type of resistance to macrolides and tetracyclines), or acquisition of a resistant form of the native, susceptible target (e.g., staphylococcal methicillin resistance caused by production of a low- affinity penicillin-binding protein).

Microbes resistance types towards antibiotics in -lactam antibiotics

Drug resistance may be acquired by mutation and selection, with passage of the trait vertically to daughter cells. For mutation and selection to be successful in generating resistance, the mutation cannot be lethal and should not appreciably alter virulence. For the trait to be passed on the original mutant or its progeny also must disseminate and replicate; otherwise, the mutation will be lost until it is "rediscovered" by some other mutant arising from within a wild-type population.

-lactam resistance mechanisms

Most -lactam antibiotics are hydrophilic and must cross the outer membrane barrier of the cell via outer membrane protein (Omp) channels, or porins. The channel has size and charge selectivity such that some Omps slow or block transit of the drug. If an Omp permitting drug entry is altered by mutation, is missing, or is deleted, then drug entry is slowed or prevented. -Lactamase concentrated between the inner and outer membranes in the periplasmic space constitutes an enzymatic barrier that works in concert with the porin permeability barrier. If the antibiotic is a good substrate for - lactamase, it will be destroyed rapidly even if the outer membrane is relatively permeable to the drug. If the rate of drug entry is slow, then a relatively inefficient -lactamase with a slow turnover rate can hydrolyze just enough drug that an effective concentration cannot be achieved. If the target (PBP, penicillin-binding protein) has low binding affinity for the drug or is altered, then the minimum concentration for inhibition is elevated, further contributing to resistance. Finally, - lactam antibiotics (and other polar antibiotics) that enter the cell and avoid -lactamase destruction can be taken up by an efflux transporter system (e.g., MexA, MexB, and OprF) and pumped across the outer membrane, further reducing the intracellular concentration of active drug.

Antibacterial drugs targets

The rational design of antibacterial agents depends upon the exploitation of a molecular structural feature found in bacteria but not found in humans. There are a number of such targets within bacteria, including the

 bacterial cell wall,  bacterial cell membrane,  bacterial protein synthesis,  bacterial nucleic acid /DNA, RNA/ synthesis. Antibacterial drug targets

Antimicrobial agents are classified based on chemical structure and proposed mechanism of action, as follows:

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1. Bacterial cell wall targets  β-lactams -Penicillins (bactericidal; inhibit cell wall cross linking) e.g., benzylpenicillin, phenoyxmethylpenicillin, ampicillin, amoxicillin, flucloxacillin, methicillin, piperacillin

-Cephalosporins (bactericidal; inhibit cell wall crosslinking) e.g., cefaclor, cefalexin, cefradine, cefuroxime, cefazolin, cefotaxime, ceftriaxone, cefoxitin, cefsulodin, ceftazidime, ceftizoxime

-Monobactams (bactericidal, β-lactam-like activity) e.g., aztreonam

-Carbapenems (bactericidal, β-lactam-like activity) e.g., imipenem

 Bacitracin (bactericidal; interrupts mucopeptide synthesis)  Vancomycin (bactericidal; interrupts mucopeptide synthesis)  Cycloserine (bactericidal; interrupts synthesis of cell wall peptides) 2. Bacterial cell membrane targets

 Polymyxins (bactericidal; disrupt bacterial membrane structural integrity) 3. Bacterial protein synthesis

 Chloramphenicol (bacteriostatic; interrupts protein synthesis at the ribosome)  Macrolides (bacteriostatic; interrupt protein synthesis at the 50S ribosome subunit) e.g., erythromycin, azithromycin, clarithromycin  Lincomycins (bacteriostatic; interrupt protein synthesis at the 50S subunit)  Aminoglycosides (bactericidal; interrupt protein synthesis at the 30S subunit) e.g., gentamicin, amikacin, kanamycin, neomycin, tobramycin  Tetracyclines (bacteriostatic; interrupt protein synthesis at the 30S subunit) e.g., tetracycline, doxycycline, minocycline 4. Bacterial nucleic acid synthesis

 Sulfonamides (bacteriostatic; inhibit bacterial folic acid synthesis)  Trimethoprim (bacteriostatic; inhibits bacterial folic acid synthesis)  Quinolones (bacteriostatic; inhibit DNA gyrase) e.g., ciprofloxacin, cinoxacin, enoxacin, norfloxacin  Rifampin (bactericidal; blocks mRNA synthesis in bacteria, inhibits RNA polymerase)

Bacterial cell wall targets

Cell wall structure

Cells are broadly classified as either prokaryotes or eukaryotes. Prokaryotic cells are found in simpler organisms, such as bacteria. They do not have a membrane enclosed nucleus, but their DNA is dispersed in the cytoplasm of the cell. All cells have a membrane, known as the plasma or cytoplasmic membrane that separates the internal medium of a cell (intracellular fluid) from its surrounding medium (extracellular fluid). Membranes also form the boundaries between the various internal regions of the cell that retain the intracellular fluid in separate compartments in the cell. Most drugs have to pass through one or more membranes to reach their site of action. Cytoplasmic membranes may also divide the interior of a cell into separate compartments. In addition to the cytoplasmic membrane, the more fragile membranes of plants and bacteria are also protected by a 101 rigid external covering known as a cell wall. The combination of cell wall and plasma membrane is referred to as the cell envelope.

Most bacteria have a well defined cell wall that covers the outer surface of the plasma membrane) of the cell. This is a rigid structure, consisting of a complex polypeptide–polysaccharide (peptidoglycan, Figures A2.2 and A2.3) matrix. Bacteria are commonly classified as being either Gram positive or Gram negative depending on their response to the Gram stain test. The cell walls of Gram- positive bacteria are about 25nm thick and consist of up to 20 layers of the peptidoglycan (Figure A2.1 (a)). In contrast, the cell walls of Gram-negative bacteria are only 2–3nm thick and consist of an outer lipid bilayer attached through hydrophobic proteins and amide links to the peptidoglycan (Figure A2.1 (b)). This lipid–peptidoglycan structure is separated from the plasma membrane by an aqueous compartment known as the periplasmic space /in which many gram negative bacteria produce β-lactamase/.

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This space contains transport sugars, enzymes and other substances. The complete structure separating the cytoplasm of the bacteria from its surroundings is known as the cell envelope. The peptidoglycans (mureins) found in both Gram-positive and Gram-negative bacteria are commonly known as mureins. They are polymers composed of polysaccharide and peptide chains, which form a single, netlike molecule that completely surrounds the cell. The polysaccharide chains consist of alternating 1–4 linked b-N-acetylmuramic acid (NAM) and b-N-acetylglucosamine (NAG) units (Figure A2.2). Tetrapeptide chains are attached through the lactic acid residues of the NAM units of these polysaccharide chains. In Gram-positive bacteria, the tetrapeptide chains of one polysaccharide chain are cross linked by pentaglycine peptide bridges from the g-amino group of lysine to the terminal alanine of the tetrapeptide chains of a second polysaccharide chain to form a net-like polymer (Figure A2.3(a)). The pentaglycinepeptide bridges occasionally contain other residues. In Gram-negative bacteria the tetrapeptide chains are directly linked by amide group (peptide bond) bridges (Figure A2.3(b)). The structure is denser than that found in the Gram-positive cell wall because the peptidoglycan chains are closer together.

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It maintains the shape and integrity of the bacteria by preventing either the swelling and bursting (lysis) or the shrinking of the bacteria when the osmotic pressure of the surrounding medium changes. The bacterial cell wall is continually being broken down by enzymes in the surrounding medium and so it is continuously being rebuilt. This rebuilding process offers a potential target for drugs since inhibition of the reconstruction processes could destroy the integrity of the bacterial cell, resulting in its death.

In general, drugs acting on microorganisms by either disrupting the structures of membranes and walls or their synthesis appear to act by:

a/ inhibitors of bacterial cell wall synthesis

 Inhibiting the action of enzymes and other substances in the cell membrane involved in the production of compounds necessary for maintaining the integrity of the cell membrane,  Inhibiting processes involved in the formation of the cell wall, resulting in an incomplete cell wall, which leads to loss of vital cellular material and subsequent death of the cell, b/ agent which increase permeability of bacterial cell wall or membrane

 Forming channels through the cell wall or membrane, making it more porous, which also results in the loss of vital cellular material and the death of the cell, and  Making the cell more porous by breaking down sections of the membrane.

Inhibitors of bacterial cell wall or cytoplasmic membrane synthesis

The successful chemotherapeutic management of any host–parasite interaction—whether viral, bacterial, or protozoan—depends upon the exploitation of biochemical differences between the host and the parasite. The greater these differences are, the better the likelihood of finding or designing drugs that exploit them and inhibit some crucial function of the parasite in order to kill it without harming the host cell. This almost utopian goal has been approximated very closely in the case of cell wall synthesis inhibitors, such as antibacterial agents, for the simple reason that a very fundamental difference exists between bacteria and mammalian cells: the former have cell walls and the latter do not. The rigid cell wall of bacteria encloses and strengthens the vulnerable cell membrane, which is subjected to considerable internal osmotic pressure. If the integrity of the cell wall is impaired, the bacterial cell will undergo breakdown (lysis) and the bacterium will perish. The antibiotics that

104 inhibit cell wall synthesis cannot find an analogous target in animal cells and are in most cases extremely nontoxic.

Peptidoglycan membrain components biosynthesis is realized by 6 stages, 3 of which are membrain dependant by Mur enzyme (A-F), which are typical for bacterial cells and can be target for antibiotics, for example phosphomicin inhibites Mur A transferase, binding with serine residue thiol groups.

Then synthesized muramyl pentapeptide forms Lipid l and Lipid ll membrane unites, which pass cell external surface /translocation/ where in transpeptidation and transglycosidation way cell wall is formed. This process also can be target for antibiotics, for example β-lactams act on transpeptidation process. Ramoplanin antibiotic /which is in clinical experimental 3 stage/ binding with Lipid ll inhibites transglycosidation.

Bacterial cell wall synthesis is a complicate process including many steps. Various investigations have elucidated many details of this process. First, the antibacterial agent has to penetrate the outer membrane of the Gram-negative bacteria, which are less susceptible to antibiotics. The β-lactam antibiotics (penicillins, cephalosporins) cross this diffusion-resistant membrane through porin channels, trimeric proteins that traverse the membrane. There are about 105 channels per bacterial cell, and their diameter is 1.2 nm. Some bacterial genera (e.g., Pseudomonas) are insensitive to most β-lactam antibiotics because the majority of their porin channels are not functional. The next hurdle the antibiotic has to surmount involves the β-lactamase enzymes in the periplasmic space, between the outer and inner membranes; these can deactivate the antibiotic (Gram-positive bacteria excrete the lactamase into the medium).

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Ionophoric antibiotics

Ionophores are substances that can penetrate a membrane and increase its permeability to ions. They transport ions in both directions across a membrane. Consequently, they will only reduce the concentration of a specific ion until its concentration is the same on both sides of a membrane. This reduction in the concentration of essential cell components of a microorganism is often sufficient to lead to the destruction of the organism. Ionophores are classified as either channel or carrier ionophores.

Channel ionophores form channels across the membrane through which ions can diffuse down concentration gradient /polyene antibiotics: amphotericine B, nistatine or polypeptides/. The nature of the channel depends on the ionophore, for example, gramicidin A /15 amino acid containing polypeptide/ channels are formed by two gramicidin molecules, N-terminus to N-terminus, each molecule forming a left-handed helix (Figure 7.1(a)). Channels increase transport of protons, Na, K, thallium.

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Nisin is a natural compound, which is produced from Lactococcus lactis microbes and now is widely used as natural conservator in low acidity food, it concerns to lantibiotics /antibiotics which contain lanthionin, 2 alanin residue connected by sulfur atom/ class. Nisin and other A type lantibiotics kill microbes /and its spores/ forming channels in their membrain, due to binding with Lipid ll.

Another novel antibiotic Mersatidin /lantibiotic B/ also inhibits cell wall action binding with Lipid ll /it is in preclinical experiments stage/.

Carrier ionophores pick up an ion on one side of the membrane, transport it across, and release it into the fluid on the other side of the membrane. They usually transport specific ions. For example, valinomycin transports K+ but not Na+ Li+ ions (Figure 7.1(b)).

These antibiotics, also called cage carriers, are doughnut-shaped molecules that displace the hydration shell of the positively charged species by several of the ether or hydroxyl oxygens of the ligand, and bind a single metal· ion in their central cavity. In other words, they form clathrate complexes with cations by virtue of multiple ion-dipole and ion-induced dipole interactions, and carry the cations in the form of a hydrophobic, ion-ionophore complex across the membrane. The 107 hydrocarbon periphery enables the complex to traverse the hydrocarbon interior of the membrane. Diffusion of these transport antibiotics is essential for their activities. Carriers form a bracelet structure stabilized by six or eight intramolecular hydrogen bonds between the amide CO and NH groups, with the ester carbonyls pointing away from the symmetry axis, all alkyl moieties on the outer boundary of the molecule. The exterior of the bracelet is hydrophobic and its interior hydrophilic.

Thus the polar interior can accommodate a nonhydrated potassium ion and surround it with an apolar bracelet. This complex can then be transported through a membrane in an energy-dependent K+–H+ exchange. The selectivity of valinomycin for K+ over Na+ is very high, the ratio being about 104:1. In this way, valinomycin will increase the K+ conductivity of lipid membranes at concentrations as low as 10−9 M. The high K+ selectivity is due to the relative ease of dehydration of this ion: with its larger diameter, the potassium ion holds hydrate water less firmly than does sodium; consequently, whereas the hydrated sodium ion does not fit the valinomycin “doughnut,” the dehydrated K+ does bind easily, with the bonding energy providing a further energy advantage for the selective reaction. A hydrated sodium ion is larger than a potassium ion with or without a hydrate envelope.

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β-LACTAM ANTIBIOTICS

The β-lactam antibiotics contain β-lactam ring (azetidinone), which represents 4-membered cyclic amide. This structural is unstable, rarely meets in nature. Lactam ring is proved to be the main component of the pharmacophore for penicillins and cefalosporines, so the term possesses medicinal as well as chemical significance. The penicillin subclass of -β lactam antibiotics is characterized by the presence of a substituted 5-membered thiazolidine ring fused to the -β lactam ring /it is fused to 6- membered thiazolidine ring in the cefalosporine subclass/. This fusion and the chirality of the β- lactam ring results in the molecule roughly possessing a "V"-shape. This drastically interferes with the planarity of the lactam bond and inhibits resonance of the lactam nitrogen with its carbonyl group. Consequently, the -β lactam ring is much more reactive and, therefore, more sensitive to nucleophilic attack when compared with normal planar amides.

Mechanism of action

The molecular mode of action of the β-lactam antibiotics is a selective and irreversible inhibition of the enzymes processing the developing peptidoglycan layer.

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Just before cross-linking occurs, the peptide pendant from the lactate carboxyl of a muramic acid unit terminates in a D-alanine-alanine unit. The terminal D-alanine unit is exchanged for a glycine unit on an adjacent strand in a reaction catalyzed by a cell wall transamidase. This enzyme is one of the penicillin biding proteins (carboxypeptidases, endopeptidase, and transpeptidase) that normally reside in the bacterial inner membrane and perform construction, repair, and housekeeping functions, maintaining cell wall integrity and playing a vital role in cell growth and division. This is β–lactams' one of the main targets. They differ significantly from bacterium, and this is used to rationalize different potency and morphologic outcomes following β –lactam attack on the different bacteria.

The cell wall transamidase uses a serine hydroxyl group to attack the penultimate D-alanyl unit, forming a covalent ester bond, and the terminal D-alanine, which is released by this action, diffuses away. The enzyme-peptidoglycan ester bond is attacked by the free amino end of a pentaglycyl unit of an adjacent strand, regenerating the active site of transpeptidase for further catalytic action and producing a new amide bond, which connects two adjacent strands together /alanine residue binds with pentaglycine/.

The three-dimensional geometry of the enzyme active site of the perfectly accommodates to the shape and separation of the amino acids of its substrate. Because the substrate has unnatural stereochemistry at the critical residues, this enzyme is not expected to attack host peptides or even other bacterial peptides composed of natural-amino acids.

The penicillins and the other β-lactam antibiotics have a structure that closely resembles that of acylated D-alanine-alanine. The enzyme mistakenly accepts the penicillin as though it were its normal substrate. The highly strained β–lactam ring is much more reactive than a normal amide moiety, particularly when fused into the appropriate bicyclic system. The intermediate acyl-enzyme complex, however, is rather different structurally from the normal intermediate in that the hydrolysis does not break penicillin into two pieces, as it does with its normal substrate. In the

110 penicillins, a heterocyclic residue is still covalently bonded and cannot diffuse away as the natural terminal n-alanine unit does. This presents a steric barrier to approach by the nearby pentaglycyl unit and, therefore, keeps the enzyme's active site from being regenerated and the cell wall precursors from being cross-linked. The resulting cell wall is structurally weak and is subject to osmotic stress. Cell lysis can result, and the cell rapidly dies.

Binding of β-lactam antibiotics to PBP-1A and PBP-1B (transpeptidases) of Escherichia coli leads to cell lysis; to PBP-2 (transpeptidase) leads to oval cells deficient in rigidity, and to inhibition of cell division; and to PBP-4 and PBP-5 leads to bacteria cell lethal effect. β–lactam antibiotics display different specificity towards different enzymes. Thus, amoxicillin and the cephalosporins bind more avidly to PBP-1, methicillin and cefotaxime to PBP-2, and mezlocillin and cefuroxime to PBP-3.

β-lactam antibiotics' chemical instability and allergenicity

The most unstable bond in the penicillin molecule is the highly strained and reactive β-lactam amide bond. This bond cleaves moderately slowly in water unless heated, but it breaks down much more rapidly in alkaline solutions to produce penicilloic acid, which readily decarboxylases to produce penilloic acid. Penicilloic acid has a negligible tendency to re-close to the corresponding penicillin, so this reaction is essentially irreversible under physiologic conditions. Because the β– lactam ring is an essential portion of the pharmacophore, its hydrolysis deactivates the antibiotic.

The origin of the allergy is a haptenic reaction with host proteins /β-lactams form antigen conjugates interacting with the protein nucleophile groups/ and the responsible bond in the drug is the β-lactam moiety, this side effect is caused by the pharmacophore of the drug and is unlikely to be over-come by molecular manipulation.

β-lactams' decomposition also takes place in acidic environment /stomach/, with side chain's amide group oxygen. Thus, benzyl penicillin remains a few minutes in the gastric juice pH conditions. Therefore, the less is preparation water solubility, the stable is the preparation.

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Resistance to β-lactam antibiotics

Today, unfortunately, resistance to β-lactam antibiotics is increasingly common and is rather alarming. It can be intrinsic and involve decreased cellular uptake of drug, or it can involve lower binding affinity to the PBPs, which is explained by their mutations in antibiotics long-term usage. Much more common, however, is the elaboration of a β-lactamase. β-lactamases are enzymes (serine proteases) elaborated by microorganisms that catalyze hydrolysis of the β-lactam bond and inactivate β-lactam antibiotics to penicilloic acids before they can reach the PBPs (Fig. 38.17). In this, they somewhat resemble the cell wall transamidase from which they may have arisen. Hydrolytic regeneration of the active site is dramatically more facile with β-lactamases than is the case with cell wall transamidase so that the enzyme can turn over many times and a comparatively small amount of enzyme can destroy a large amount of drug. With Gram-positive bacteria, such as staphylococci, the β-lactamases usually are shed continuously into the medium and meet the drug outside the cell wall. Thus, they are biosynthesized in significant quantities. With Gram-negative bacteria, a more conservative course is followed. Here, the β-lactamases are secreted into the periplasmic space between the inner and outer membrane, so although still distal to the PBPs, they do not readily escape into the medium and need not be resynthesized as often. Numerous β-lactamases with various antibiotic substrate specificities are now known. Elaboration of β-lactamases is induced by the presence of β-lactam antibiotics.

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β-lactamase enzyme inhibitors

Nowadays, β-lactams combinations with β–lactamase inhibitors are widely used for increasing β- lactams stability. β–lactamase inhibitors are clvulanic acid and sulbactam.

Clavulanic acid is a mold product with only weak antibacterial activity, but it is an excellent irreversible inhibitor of most β-lactamases. It is believed to active site serine by mimicking the normal substrate. Hydrolysis occurs with some β-lactamases, but in many cases, subsequent reactions occur that inhibit the irreversibly. This leads to its classification as a mechanism-based inhibitor (or so-called suicide substrate). When clavulanic acid is added to ampicillin and amoxicillin preparations, the potency against β-lactamase-producing strains is markedly enhanced. Clavulanic acid and amoxicillin combination (augmentin, amoxiclav) is widely used in medicine.

Sulbactam is prepared by partial chemical synthesis from penicillins. The oxidation of the sulfur atom to a dioxide greatly enhances the potency of sulbactam, irreversibly binding with β-lactamase enzyme. The combination of sulbactam and ampicillin (Unasyn) is now clinically popular.

SAR for Penicillins

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The chemical substituents to the penicillin nucleus can greatly influence the stability of the penicillins as well as the spectrum of activity.

 The substitution of a side-chain R group on the primary amine with an electron-withdrawing group decreases the electron density on the side-chain carbonyl and protects these penicillins, in part, from acid degradation. This property has clinical implications, because these compounds survive passage through the stomach better and many can be given orally for systemic purposes /phenoxymethyl penicillin, amoxicillin, oxacillin/.

Benzylpenicillin Phenoxymethilpicillin

Amoxicillin

Semi-synthetic penicillinase-resistant oral penicillins

 The more lipophilic the side chain of penicillin, the more serum protein bound is the antibiotic. This has some advantages in terms of protection from degradation, but it does reduce measurably the effective bactericidal concentration of the drug in whole blood.  Stability of the penicillins toward β-lactamase is influenced by the bulk in the acyl group attached to the primary amine. β-lactamases are much less tolerant to the presence of steric hindrance near the side-chain amide bond than are the penicillin binding proteins. Thus, importing metoxy group in ortho position toward the amide group, preparation possesses stability toward β- lactamase.

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 Replacing one of the metoxy groups from ortho into para position and also importing methylene bridge between aromatic ring and amide group, lead to sensitivity increase toward β-lactamase (meticillin, nafcillin, dicloxicillin, oxacillin, cloxicillin). Semi-synthetic penicillinase-resistant parenteral penicillins

SAR for cephalosporines

As with the penicillins, various molecular changes in the cephalosporin can improve in vitro stability, antibacterial activity, and stability toward β-lactamases.

 Changes of the side chain substitutes lead to spectrum change. This gives chance to differentiate 4 generation of cephalosporines, which are differed from each other by spectrum broad.  The addition of an amino and a hydrogen to the a and a' position, respectively, results in a basic compound that is protonated under the acidic conditions of the stomach (as in penicillins). The ammonium ion improves the stability of the β-lactam of the cephalosporin, leading to orally active drugs (cephalexin, cephradine).

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First-generation cephalosprines

 The 7 β-amino group is essential for antimicrobial activity (X=H), whereas replacement of the hydrogen at C-7 (X=H) with an alkoxy (X=OR) results in improvement of the antibacterial activity of the cephalosporin. Within specific cephalosporin derivatives, the addition of a 7a methoxy also improves the drugs stability toward β-lactamase and the preparations become active to the some G- negative microbes.

Second-generation cephalosprines

 More activity is observed in case of S atom presence in the ring, though replacing S atom by oxygen atom increases stability toward β-lactamase.  In a study examining the stability of cephalosporins toward β-lactamase, it was noted that the groups' spatial orientation influences on β-lactamase resistance. Thus L-isomer of the side chain substituent is 30- to 40-fold more stable than the D-isomer.

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Third-generation cephalosprines

 The addition of a methoxyoxime to a position increased stability nearly 100-fold (cefuroxime, ceftazidime, cefixime).  The Z-oxime was as much as 20,000-fold more stable than the E-oxime.

 Importing 5-membered heterocyclic ring in C3 carbon (Z) activity increases. Thus importing quaternary N-methyl pyrrolidine ring lead to creation of 4 generation cephalosporin representative cefepime, which possesses high permeability of G- bacteria wall.

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TETRACYCLINES

Naphthacene

The tetracycline family is widely, but not intensively, used in office practice. This family of antibiotics is characterized by a highly functionalized, partially reduced naphthacene (four linearly fused, 6-membered rings) ring system from which both the family name and the numbering system are derived. They possess a number of adverse effects, although most of them are annoying rather than dangerous. The advent of other choices and the high incidence of resistance that has developed have greatly decreased their medicinal prominence in recent years. Presently, they are recommended primarily for use against rickettsia, chlamydia, mycoplasma, anthrax, plague, and helicobacter organisms. A significant number of semi synthetic molecules derived from the antibacterial tetracyclines have shown potential activity in other therapeutic areas, such as antimetastasis, antitumor, anti-inflammatory, antiarthritic, antifungal, antineurotoxic, and antiperiodontal diseases, but discussion of these potential uses is beyond the scope of this chapter.

Natural tetracyclines were isolated from members of Streptomyces family. Some tetracyclines have been obtained by semi synthetic way, some of them have been totally synthesized.

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Chemical Properties

The tetracyclines have complex stereo chemical structure, they contain 5-6 asymmetric carbon atom.

The tetracyclines are yellow, light-sensitive compounds. They are amphoteric substances with three pKa values revealed by titration (2.8-3.4, 7.2-7.8, and 9.1-9.7). The basic function is the C-4-α- dimethylamino moiety. Commercially available tetracyclines generally are administered as comparatively water-soluble hydrochloride salts. The conjugated phenolic enone system extending from C-10 to C-12 is associated with the pKa at approximately 7.5, whereas the conjugated trione system extending from C-1 to C-3 in ring A is nearly as acidic as acetic acid (pka- 3).

These resonating systems can be drawn in a number of essentially equivalent ways with the double bonds in alternate positions. The formulae normally given are those settled on by popular convention.

Chelation

Chelation is an important feature of the chemical and clinical properties of the tetracyclines. The acidic functions of the tetracyclines are capable of forming salts through chelation with metal ions. The salts of polyvalent metal ions, such as Fe2+, Ca2+, Mg2+, and AI3+, are all quite insoluble at neutral pH. This insolubility not only is inconvenient for the preparation of solutions but also interferes with blood levels on oral administration. Consequently, the tetracyclines are incompatible with coadministered, multivalent ion-rich antacids and with hematinics, and concomitant consumption of daily products rich in calcium ion also is contraindicated. Further, the bones, of which the teeth are the most visible, are calcium-rich structures at nearly neutral pHs and so accumulate tetracyclines in proportion to the amount and duration of therapy when bones and teeth are being formed.

Because the tetracyclines are yellow, this leads to a progressive and, essentially, permanent discoloration in which, in advanced cases, the teeth are even brown.

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The intensification of discoloration with time is said to be a photochemical process. This is cosmetically unattractive but does not seem to be deleterious except in extreme cases, when so many antibiotics is taken up that the structure of bone is mechanically weakened. To avoid this, tetracyclines are not normally given to children once they are forming their permanent set of teeth (age, 6-12 years). In severe cases, the teeth can be treated with dilute HCl solution to dissolve away the colored antibiotic. This also significantly erodes the mineral matrix of the teeth, however, and must be repaired by plastic impregnation. People naturally prefer to avoid this heroic and expensive process. When concomitant oral therapy with tetracyclines and incompatible metal ions must be done, the ions should be given 1 hour before or 2 hours after the tetracyclines. Additionally, tetracyclines are painful on 1M injection. This has been attributed, in part, to formation of insoluble calcium complexes. To deal with this, the injectable formulations contain ethylenediaminetetraacetic acid and are buffered at comparatively acidic pH levels where chelation is less pronounced and water solubility is higher.

Epimerization

The α-stereo orientation of the C-4 dimethylamino moiety of the tetracyclines is essential for their bioactivity. The presence of the tricarbonyl system of ring A allows enolization involving loss of the C-4 hydrogen. Reprotonation can take place from either the top or bottom of the molecule. Reprotonation from the top of the enol regenerates tetracycline. Reprotonation from the bottom, however, produces inactive 4-epitetracycline. At equilibrium, the mixture consists of nearly equal amounts of the two diasteromers. Thus, old tetracycline preparations can lose approximately half their potency in this way. The epimerization process is most rapid at approximately pH 4 and is relatively slower in the solid-state.

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Dehydration.

Most of the natural tetracyclines have a tertiary benzyl hydroxyl group at C-6. This function has the ideal geometry for acid-catalyzed dehydration involving the C-5a a-oriented hydrogen (antiperiplanar trans). The resulting product is a naphthalene derivative, so there are energetic reasons for the reaction proceeding in that direction. C-5a, 6-anhydrotetracycline is much deeper in color than tetracycline and is biologically inactive.

Discolored old tetracyclines are suspect and discarded. Not only can inactive 4-epitetracyclines dehydrate to produce 4-epianhydrotetracyclines, anhydrotetracycline also can epimerize to produce the same product. This degradation product is toxic to the kidneys and produces a Fanconi-like syndrome that, in extreme cases, has been fatal. Commercial samples of tetracyclines are closely monitored for the presence of 4-epidehydrotetracycline and injuries from this cause are now fortunately, rare. Those tetracyclines, such as minocycline and doxycycline, which have no C-6- hydroxyl groups, cannot undergo dehydration and, therefore, are completely free of this toxicity.

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Cleavage in Base

Another untoward degradation reaction involving a C-6-hydroxyl group is cleavage of the C ring in alkaline solutions at or above. The lactonic product, an isotetracycline, is inactive. The clinical impact of this degradation under normal conditions is uncertain.

Phototoxicity

Certain tetracyclines, most notably those with a C-7-chlorine, absorb light in the visible region, leading to free radical generation and, potentially, cause severe erythema to sensitive patients on exposure to strong sunlight. Patients should be advised to be cautious about such exposure for at least their first few doses to avoid potentially severe sunburn. This effect is comparatively rare with most currently popular tetracyclines.

Mechanism of Action

The tetracyclines of clinical importance interfere with protein biosynthesis at the ribosomal level, leading to bacteriostasis.

Tetracyclines bind to rRNA in the 30S subparticle with the possible cooperation of a 50S site by a process that remains imprecisely understood despite intensive study. There is more than one binding site, but only one is believed to be critical for its action. The points of contact with the rRNA are 122 those associated with antibiosis with the puzzling exception of the dimethylamino function. Studies have suggested that the tetracyclines bind to the 16S rRNA via the functional groups located at the 1- and 10- to 12 α-positions (referred to as the southern face of the tetracycline) and at the 2- and 3- positions (referred to as the eastern face of the tetracycline). The dimethylamino function is known to be essential for activity but does not appear to bind in the x-ray pictures that are presently available. Once the tetracycline binds, it inhibits subsequent binding of aminoacyltransfer-RNA to the ribosomes, resulting in termination of peptide chain growth. Newer analogues of the tetracyclines suggest that substitution on the western and northern faces of the tetracycline are allowed as indicated by the newest glycylcyclines.

The more lipophilic tetracyclines, typified by minocycline, also are capable of disrupting cytoplasmic membrane function, causing leakage of nucleotides and other essential cellular components from the cell, and have bactericidal properties. The more lipophilic tetracyclines enter bacterial cells partly by passive diffusion and the more water-soluble members partly through water- lined protein porin routes, perhaps assisted by the formation of highly lipophilic calcium and magnesium ion chelates.

Deeper passage, however, through the inner cytoplasmic membrane is an energy-requiring active process, suggesting that the tetracyclines are mistaken by bacteria as food.

SAR

 The pharmacophoric group with full biological activity is the partially synthetic sancycline, which contains the structural requirements for chelation to be as important for the transport of tetracyclines into bacterial cells as it is to the inhibition of protein biosynthesis once the drug is within the cells.

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 The linear arrangement of the four rings is a prerequisite for antibiotic activity. Relation of the configuration of the chiral centers C-4, C-4 α and C-12 is essential for activity, whereas the configurations at C-5α and c-6 may be altered. The BCD phenoldiketone system is essentially planar, but the AB ring system can undergo conformational changes. At physiological pH all the tetracyclines have identical conformations. All partial or otally synthetic derivatives having fewer than four rings are inactive. Opening of the rings or aromatization of additional rings results in inactive compounds.

 The presence of the 4-dimethylamino moiety is essential for the formation of the zwitterions and therefore for optimum distribution within the body and for in vivo activity; removal of this moiety results in a substantial loss of activity; furthermore, this moiety must retain the stereochemistry of the natural tetracyclines.  In the 2-amide moiety, only the carbonyl group is essential for activity; one amide hydrogen atom can be replaced without loss of activity.  The hydrophobic region of the molecule from C-5 to C-9 can be altered in various ways as long as there is no interference with the above-stated essential features. Modifications of C-6 and C-7 result in derivatives having greater chemical stability, increased antibiotic activity, and improved pharmacokinetics. Such is the case with doxycycline and minocycline, known as 6- deoxytetracyclines, because they lack the 6-hydroxy group and therefore do not undergo the degradation reactions.

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SULFONAMIDES

These compounds consist of the following structural units:

 benzene ring with only two substituents oriented para to each other,  4-amino group (or a group such as azo, nitro, or substituted amino that in vivo yields 4-amino group),  the singly substituted l-sulfonamide group. They are, therefore, derivatives of sulfanilic acid, which exists both in molecular form and in ionized form:

Sulfonamide nitrogen and anilino nitrogen are designated N1 and N4, respectively. The group p-

NH2-C6H4S02- is called sulfanilyl; the group p-NH2-C6H4-S02NH- sulfanilamido; and the group p-NH2-

S02 - sulfonamide.

Sulfonamide is a generic term that denotes three different cases:

1. Antibacterial agents that are aniline-substituted sulfonamides (the "sulfanilamides”):

Streptocide

2. Prodrugs that react to generate active sulfanilamides (i.e., sulfasalazine):

Sulfasalazine

3. Non aniline sulfonamides (i.e., mafenide acetate):

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O O S NH

H2N O CH3 Mafenide acetate

When the first sulfonamides and their antimicrobial activity were discovered 20000 compounds were synthesized based on their structure. Structural changes include the followings: benzene ring isoster replacement, substitutions insertion into the ring, 2 para positions functional groups substitution, etc. In the result of this not only effective antimicrobial drugs were obtained, but also

1. antibacterial sulfonamides, 2. leprostatic preparations /sulfons/, 3. diuretics /carboanhidraze inhibitors, thiazide diuretics/, 4. hypoglycemic agents /sulfonyl urine derivatives/, 5. anti malarial agents, 6. anti thyroidal preparations, 7. agents for podagra treatment /probenecide/. Classification

Sulfonamides can be classified according to their chemical structure, action spectrum, duration, and place. According to their action place they are:

 systemic  intestinal  acting in the urine system  local Intestinal sulfonamides are used for intestinal infections. The 4-amino group is substituted by hydrophilic moieties: phthalyl (phthalazole) or succinyl, obtained sulfanylamides are very hydrophilic and almost not absorbed from the GIT /gastrointestinal tract/ (about 5%). This reaches a high concentration in the colon lumen, where bacterial hydrolysis slowly releases the parent sulfonamide.

O S HN S NH

C O N

C O

HO Phthalazole

Salazopyridazine can be a prodrud example, which in per os intake can’t be absorbed from the intestines, but hydrolyzed, releasing sulfapyridazine and 5-amino salicylic acid /Mesalamine/, which has anti inflammatory activity.

O N H2N COOH O N S NH OCH3 S N O N N HO N O H OH

O C H 2 N +

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Salazopyridazine Sulfapyridazine Mesalamine

In contrast to the intestinal sulfonamides, urinary sulfonamides are very lipophilic, they are rapidly absorbed in the intestines, but slowly excreted from the kidneys and thus reach a high concentration there: sulfacetamide, sulfadimethoxine, sulfamethoxazole (drugs with corresponding pKa values), (see crystalluria and pka).

Mechanism of Action

In therapeutic doses sulfonamides possess bacteriostatic activity.

Sulfonamides are structurally similar to p-aminobenzoic acid. This acid is a vitamin to bacteria, which require it for the synthesis of folic acid. Tetrahydrofolic acid, a derivative of folic acid, is a cofactor in the synthesis of thymidine, purines, and finally, DNA.

Mammals also require folic acid as an essential growth factor, but they get it from food intake. At physiological pH, folic acid exists as a dianion, which cannot cross the bacterial cell wall by passive diffusion, because its negative charges are either attracted or repelled by opposite or equal charges of the bacterial wall. Therefore, an energy-requiring active transport mechanism should be involved.

NH O N O N N H - O O

H2N N N O- Folic acid

However, with the exception of Streptococcus aecium, bacteria lack this mechanism. For this reason, these bacteria have to synthesize folic acid de novo from p-aminobenzoic acid, and consequently, this process is inhibited by sulfonamides, producing a bacteriostatic effect.

Two bacterial enzyme systems capable of performing the synthesis of folic acid have been described by Weisman and Brown: the first one, called dihydropteroate synthase, catalyzes the synthesis of a precursor of folic acid, namely, dihydropteroic acid, through condensation of p- aminobenzoic acid with a pteridine derivative; with the subsequent addition of dihydropteroic acid to glutamic acid the biosynthesis of folic acid is completed; and the second one catalyzes the synthesis of folic acid itself by direct coupling of p-aminobenzoyl glutamate with a pteridine derivative. Both routes of folic acid biosynthesis can be antagonized by sulfonamides.

Shefter and co-workers proposed that N' substituents in sulfonamides may play the role of competing for a site on the enzyme surface reserved for the glutamate residue in p-aminobenzoic acid-glutamate.

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There is another theory (Vood’s theory), which explains mechanism of sulfanyl amides. In some bacteria’s, sulfanyl amide, due to it structural similarity, is included in the folic acid synthesis, instead of PABA, forming ‘’false folic acid’’, which can’t take part in later biosynthesis process (condensation with glutamic acid). This false metabolite also is enzyme inhibitor. Thus, sulfanylamides act when PABA content ends in bacteria and synthesis of nucleonic acid becomes impossible. Bacteria, which can get folic acid from external media, are stable toward sulfonamides.

Inhibition by sulfonamides of the enzyme systems involved in folic acid biosynthesis can be reversed by addition of a small amount of PABA: 10-4 M of PABA to 1M of a sulfonamide. Since PABA competes with sulfa drugs, local anesthetics with a p-aminobenzoic moiety-procaine, for example, should not be applied during treatment with sulfonamides, because esterases split these local anesthetics, yielding PABA.

Now sulfanyl amides are used with DHFR inhibitors /trimetoprim/.

DHFR is widely studied for creation of antimicrobial and anticancer drugs. For example, Metotrexate and its derivatives were created on the base of these studies. But Metotrexate is very toxic for using as an antibiotic, and in 1969 trimetoprim was created which inhibites DHFR and prevent FAH4 synthesis.

This enzyme is present both in human and microbes, thus preparation has an influence on human enzyme too. But the affinity of trimethoprim for bacterial dihydrofolate reductase is about 50,000 times greater than for the human enzyme. The most important result of this sequential enzymatic inhibitior seems to be the blockade of thymidine synthesis, usually producing a bactericidal effect. The use of two chemotherapeutic agents simultaneously should also diminish the rate of emergence of resistant bacterial strains.

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It is, however, most commonly used in a 1:5 fixed concentration ratio with the sulfonamide sulfamethoxazole (Bactrim, Septra). This combination is not only synergistic in vitro but also is less likely to induce bacterial resistance than either agent alone. It is rationalized that microorganisms not completely inhibited by sulfamethoxazole at the pteroate condensation step will not likely be able to push the lessened amount of substrates that leak past a subsequent blockade of dihydrofolate reductase. Thus, these agents block sequentially at two different steps in the same essential pathway, and this combination is extremely difficult for a naive microorganism to survive.

Pairing these two particular antibacterial agents was based on pharmacokinetic factors and convenient availability. For such a combination to be useful in vivo, the two agents must arrive at the necessary tissue compartment where the infection is at the correct time and in the correct ratio. In this context, the optimum ratio of these two agents in vitro is 1:20. Of all the combinations tried, sulfamethoxazole came closest to being optimal for trimethoprim. Administration of a 1:5 combinations of the two drugs orally produces the desired 1:20 ratio in the body once steady state is reached.

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SAR

Various physical and chemical parameters have been correlated with chemotherapeutic activity of sulfonamides: pKa' protein binding, and electronic charge distribution, among others. Unfortunately, no single parameter can explain the action of sulfonamides.

pKa

Bell and Roblin postulated that the antibacterial activity of sulfonamides is related to pKa-. Maximal activity would be found in those having a pK" value between 6.0 and 7.5 pK is calculated from the following equation:

When the sulfonamide is 50% dissociated, [HA]/[A -] = 1. Since log1 = 0, in this case pKa = pH. According to some authors, this is the pKa for maximal activity, because sulfonamides penetrate the cell wall as undissociated molecules but act in ionized forms.

However, the hypothesis that acidity or ionization alone explains the degree of activity of sulfonamides does not apply to all series of these drugs. Many sulfonamides lying outside the values of pKa (6.0 to 7.5) postulated by Bell and Roblin as being connected with maximal activity are potentially capable of high activities, especially those having electron withdrawing substituents on an N'-heterocyclic ring.

The functional group that differs in the two molecules is the carboxyl of PABA and the sulfonamide moiety of sulfanilamide. The strong electron-withdrawing character of the aromatic SO2 130 group makes the nitrogen atom to which it is directly attached partially electropositive. This, in turn, increases the acidity of the hydrogen atoms attached to the nitrogen so that this functional group is slightly acidic (pka =10.4)

The pka of the carboxyl group of PABA is approximately-6.5. It was soon found, following a crash synthetic program, that replacement of one of the NH2 hydrogen by an electron-withdrawing heteroaromatic ring was not only consistent with antimicrobial activity but also greatly acidified the remaining hydrogen and dramatically enhanced potency. The poor water solubility of the earliest sulfonamides led to occasional crystallization in the urine (crystalluria) and resulted in kidney damage, because the molecules were un-ionized at urinary pH values. It is still recommended to drink increased quantities of water to avoid crystalluria when taking certain sulfonamides. This form of toxicity is now comparatively uncommon with the more important agents used today, however, because these agents form sodium salts that are at least partly ionized and, hence, reasonably water soluble at urinary pH values. They are poorly tolerated on injection, however, because these salts are corrosive to tissues.

Structural variation among the clinically useful sulfonami des is restricted primarily to installation of various heterocyclic aromatic substituents on the sulfonamide nitrogen.

N Ag+ - H2N SO2N

Crystalluria and the pKa

Despite the tremendous ability of sulfanilamide to effect cures of pathogenic bacteria, its benefits were often offset by the propensity of the drug to cause severe renal damage by crystallizing in the kidneys. Sulfanilamides and their metabolites (usually acetylated at N4) are excreted almost entirely in the urine. The pKa of the sulfonamido group of sulfanilamide is 10.4, so the pH at which the drug is 50% ionized is lOA. Obviously, unless the pH is above the pKa, little of the water-soluble salt is

131 present. Because the urine is usually about pH 6 (and potentially lower during bacterial infections), essentially all of the sulfanilamide is in the relatively insoluble,

nonionized form in the kidneys. The sulfanilamide coming out of solution in the urine and kidneys causes crystalluria.

Early approaches to adjusting the solubility of sulfanilamide in the urine were

1. Greatly increasing the urine flow. During the early years of sulfonamide use, patients taking the drugs were cautioned to"force fluids." The idea was that if the glomerular filtration rate could be increased, there would be less opportunity for seed crystals to form in the renal tubules.

2. Increasing the pH of the urine. The closer the pH of the urine is to 10.4 (for sulfanilamide itself), the more of the highly watersoluble salt form will be present.

Oral sodium bicarbonate sometimes was, and occasionally still is, given to raise urine pH. The bicarbonate was administered before the initial dose of sulfanilamide and then prior to each successive dose.

3. Preparing derivatives of sulfanilamide that have lower pK. values, closer to the pH of the urine. This approach has been taken with virtually all sulfonamides in clinical use today. Examples of the pKa values of some ionizable sulfonamides are shown in Table 8.

4. Mixing different sulfonamides to achieve an appropriate total dose. The solubilities of the sulfonamides are independent of each other, and more of a mixture of sulfanilamides can stay in water solution at a given pH than can a single sulfonamide. Hence, trisulfapyrimidines, USP (triple sulfa), contain a mixture of sulfadiazine, sulfamerazine, and sulfamethazine.

Electron-Charge Distribution

Several electronic parameters show significant relationships to antibacterial action of sulfonarnides. For example, there is a qualitative relationship between activity and the formal π charge on N1: the greater this charge, the greater the biological activity. Thus, electro-acceptor group presence not only assists sulfonamides safety for kidneys, but also increases activity.

Distribution factor

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Sulfonamides lipophilic and hydrophilic properties are very important for antibacterial and pharmacokinetic properties. Hydrophilic properties are necessary for drug excretion, without crystalluria formation, and for intestinal antibiotic creation.

Metabolism

Sulphanylamides less undergo metabolism. They are metabolized in liver by N-acetylation /due to

NH2 group/ or by glucuronidation, forming inactive metabolites. Sulphanylamides and its metabolites are excreted via urine.

Resistance

Generally resistance develops in G- microbs, either PABA synthesis increases, or decreases DHPS sensitivity toward sulfanylamides, or attack mechanisms are activated. If microb is resistant to one sulfanylamides as a rule it is resistant to other preparations.

Key points

• It must contain PABA structural analog representing p-NH2-C6H4-SO2-NH- sulfanilamide part for preparation antimicrobial activity. • p-NH2 group must be not substituted for the activity, substituted derivatives or azo or nitro derivatives are prodrugs and must form free amino group in the body. • Prodrugs creation mainly has a goal to change pharmacokinetic properties and to obtain drugs acting in the certain place of the body. • Substitutions in aromatic ring must be in para-position to each other, substitutions insertion in the other positions decreases activity.

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• SO2-NH- should have acidic properties, the most active are preparations, which pKa value is 5.0-7.0. • Insertion of electroacceptor substitutitons increases nitrogen acidic properties, decreases pKa and compound becomes more active, besides decreases risk of crystalluria formation. • Heterocycles are mainly served as an electroacceptor substituent; more simple groups insertion leads to either less active or more toxic compounds creation.

QUINOLONES

Quinolones represent synthetic antimicrobial preparations, which have N-1-alkyl-3- carboxypyrid-4-on ring condensed with other aromatic ring, which can have different substituents.

The first quinolone to be marketed (in 1965) was nalidixic acid. Nalidixic acid and cinoxacin are classified as first-generation quinolones based on their action spectrum and pharmacokinetic properties. While still available, they are considered to be minor urinary tract disinfectants that are effective primarily against certain susceptible Gram-negative bacteria. Thus, the quinolones were of little clinical significance until the discovery that the addition of a fluoro group to the 6-position of the basic nucleus greatly increased the biological activity. Brought to the market in 1986, norfloxacin, the first of the second-generation quinolones /or fluoroquinolones/, has a broad spectrum and equivalent in potency to many of the fermentation derived antibiotics.

Following its introduction, intense competition ensued, more than a thousand second, third-, and fourth-generation analogues have now been made. Ciprofloxacin and levofloxacin dominate the

134 worldwide fluoroquinolone market. It should be noted that the more recent quinolones also are referred to as the fluoroquinolones these agents are now an important class of antimicrobial agents.

Fluoroquinolones frequently are called antibiotics, but they represent synthetic compounds and don’t meet in nature. According to their chemical structure they are divided into:

 naphthiridines /nalidixic acid, enoxacin/, which are 2 pyridine rings condensation result,  cinolines /cinoxacin/ instead of pyridine ring they contain pyridazine ring,  qinolines /norfloxacin, ciprofloxacin, lomefloxacin, etc./ pyridine ring is condensed with an aromatic ring.

Action mechanism 135

The quinolones are rapidly bactericidal, they inhibit DNA synthesis, largely as a consequence of inhibition of DNA gyrase and topoisomerase IV, key bacterial enzymes that dictate the conformation of DNA /tertiary structure/ and for replication, transcription. The procariots /bacteria/, e.g. E. coli chromosome is a single, circular molecule approximately 1 mm in length, whereas the cell is only 1-3 nm long. Thus, the DNA molecule must be dramatically compacted in a conformationally stable way so that it can fit. Using the energy generated by adenosine triphosphate (ATP) hydrolysis, the molecule is progressively wound about itself in a positive super coil. In the absence of ATP, the process is reversed, relaxing the molecule. It also must be partially unwound so that the cell has access to the genetic information that it contains. This requires reversible conformational changes so that it can be stored properly, unwound, replicated, repaired, and transcribed on demand. These enzymes alter the conformation of DNA by catalyzing transient double-strand cuts staggered by four base pairs, passing the uncut portion of the molecule through the gap, and resealing the molecule back together. In this way, DNA gyrase alters the degree of DNA twisting by introducing negative DNA super coils, releasing tensional stress in the molecule. According to the suggestions, quinolone molecules bind with DNA, forming complex, which consist of 4 quinolones molecules, DNA double- chain and DNA gyrase; this complex is formed when DNA gyrase splits DNA chain.

Bacterial DNA gyrase /it doesn’t exist in eukaryotes cells/ is a tetrameric enzyme consisting of two A and two B subunits, encoded by the gyrA and gyrB genes. Bacterial strains resistant to the quinolones have been identified, with decreased binding affinity to the enzyme because of amino acid substitution in the A and B subunits resulting from mutations in either gyrA57 or gyrB58 genes. The highly polar quinolones are believed to enter bacterial cells through densely charged porin channels in the outer bacterial membrane. Mutations leading to altered porin proteins can lead to decreased uptake of quinolones and cause resistance. Also, there is evidence for energy-dependent efflux of quinolones by some bacterial species. A quantitative structure-activity relationship (QSAR) study of bacterial cellular uptake of a series of quinolones revealed an inverse relationship of uptake versus log P (a measure of lipophilicity) for Gram-negative bacteria, on the one hand, but a positive correlation of quinolone uptake to log P in Gram-positive bacteria, on the other. This result probably reflects the observed differences in outer cell wall structures of Gram-negative and Gram-positive bacteria.

DNA topoisomerase IV decatenates (unties) enchained daughter DNA molecules produced through replication of circular DNA. Inhibition of DNA gyrase and topoisomerase IV makes a cell's DNA inaccessible and leads to cell death. Different quinolones inhibit these essential enzymes to different extents. Topoisomerase IV seems to be more important to some Gram-positive organisms 136 and DNA gyrase to some Gram-negative organisms. Humans shape their DNA with a topoisomerase II, an analogous enzyme that does not bind quinolones at normally achievable doses, so the quinolones of commerce do not kill host cells.

SAR

The structural features of the quinolones strongly influence the antimicrobial and pharmacokinetic properties of this class of drugs. Structure-activity studies have shown that the 1,4- dihydro-3-carboxy-4-pyridone ring is essential for antibacterial activity. The pyridone /pyridine-4- on/ system must be condensed with an aromatic ring.

Apparently, the carboxylic acid and the ketone are involved in binding to the DNA/DNA gyrase enzyme system.

 Reduction of the 2,3-double bond or the 4-keto group inactivates the molecule.  Substitution at C-2 interferes with enzyme-substrate complexation.  Fluoro substitution at the C-6 position greatly improves antimicrobial activity /action spectrum as well/ by increasing the lipophilicity of the molecule, which in turn improves the drugs penetration through the bacterial cell wall. Additionally, C-6 fluoro increases the DNA gyrase inhibitory action. In resalt 2nd generation fluoroquinolons /norfloxacin, ciprofloxacin/ were obtained.  An additional fluoro group at C-8 further improves drug absorption and half-life /longer duration/, but also may increase drug-induced photosensitivity /undesirable effect/.  Heterocyclic substitution at C-7 improves the spectrum of activity especially against Gram- negative organisms, e.g. 3-aminopyrrolidinyl substituent at C-7 enhances activity against P. aeruginosa. The piperazinyl (as in ciprofloxacin) and pyrrolidinyl (as in moxifloxacin) represent the most significant antimicrobial improvement. Unfortunately, the piperazinyl group at C-7 also increases binding to central nervous system (CNS) Y-aminobutyric acid (GABA) receptors, which accounts for CNS side effects. Alkyl substitution on the piperazine (lomefloxacin, ofloxacin, sparfloxacin) is reported to decrease binding to GABA.  The alkyl substitution /methyl, ethyl, cyclopropyl/ at N-l appears to broaden activity of the quinolones to include activity against atypical bacteria, including Mycoplasma, Chlamydia, and Legionella species.  Aryl substitute insertion in that position increases activity; 2,4-diflourphenyl substitunt is the most optimal for the activity.

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 The introduction of a third ring to the nucleus of the quinolones gives rise to the active preparations /ofloxacin/. Additionally, ofloxacin has an asymmetric carbon at the C-3' position. The S- (-)-isomer (levofloxacin) is twice as active as ofloxacin and 8- to 128-fold more potent than the R- (+)-isomer resulting from increased binding to the DNA-gyrase.

Acid-basic properties

The antibacterial quinolones can be divided into two classes on the basis of their dissociation properties in physiologically relevant conditions. The first class, represented by nalidixic acid, oxolinic acid, and cinoxacin, possesses only the 3-carboxylic acid group as an ionizable functionality. The pKa values for the 3-carboxyl group in nalidixic acid and other quinolone antibacterial drugs fall in the range of 5.6 to 6.4. These comparatively high pKa values relative to the pKa of 4.2 for benzoic acid are attributed to the acid-weakening effect of hydrogen bonding of the 3-carboxyl group to the adjacent 4-carbonyl group.

The second class of antibacterial quinolones embraces the broad-spectrum fluoroquinolones (namely, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, lomefloxacin, and sparfloxacin), all of which possess, in addition to the 3-carboxylic acid group, basic piperazino functionality at the 7 position and a 6-fluoro substituent. The pKa values for the more basic nitrogen atom of the piperazino group fall in the range of 8.1 to 9.3. At most physiologically relevant pH values, significant dissociation of both the 3-carboxylic acid and the basic 7-(l-piperazino) groups occurs, leading to significant fractions of zwitterionic species.

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The tendency for certain fluoroquinolones (e.g., norfloxacin and ciprofloxacin) in high doses to cause crystalluria in alkaline urine is, in part, due to the predominance of the comparatively less soluble zwitterionic form.

Side effects

As a precaution, these drugs are not used casually in children younger than 18 years or in sexually active females of childbearing age. Some effective preparations aren’t used in medicine due to their toxic effects, e.g. temofloxacin (causes hemolysis, thrombocytopenia), grepafloxacine (cardiotoxic), chlorine substituted derivatives in 8 position (clinafloxacin, sitafloxacin) were very phototoxic.

CNS

Derivatives, which have substituent in 7 position, especially 1-piperidine, 3-amino-1pyrrolidine or other basic substitute (piperazine) have antagonistic properties toward central GABA receptors (sleep disorder, trembling, etc.).

Phototoxic influence

Several of the quinolones produce mild to severe photosensitivity. A C-8 halogen appears to produce the highest incidence of photosensitivity via singlet oxygen and radical induction. Lomefloxacin has been reported to have the highest potential for producing phototoxicity. Substitution of a methoxy group at C-8 has been reported to reduce the photosensitivity (gatifloxacin).

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Incompatibility

Chemical incompatibility common to all the quinolones involves the ability of these drugs to chelate (Ca2+, Mg2+, Zn2+, Fe2+, Al3+, etc.) polyvalent metal ions resulting in decreased solubility and reduced drug absorption. Chelation occurs between the metal and the 3-carboxylic acid and 4- keto groups.

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ANTITUBERCULAR AGENTS

Isonicotinic acid derivatives

Isonicotinic acid derivatives are synthetic antimicrobial agents which display bactericidal activity towards Mycobacterium tuberculosis. Thus, bactericidal activity is basically shown towards the microorganisms which are in division stage and bacteriostatic towards microbes, being in a rest stage. Derivatives of this group are isoniazid, ftivazide, methazid and isonicotinic acid thioamide derivatives: ethionamide and protionamide.

Isonicotinic acid derivatives

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Isoniazid Ftivazide

Isonicotinic acid thioamide derivatives

Ethionamide Protionamide

The last 2 preparations are antitubercular agents of the second group. These compounds show high activity, but quickly cause tolerance and have expressed side effects. They are basically used in case of microbes’ resistance to the preparations first group.

Action mechanism

The action mechanism is completely obscure; however, it is known that isoniazid is a prodrug, which is activated in an oxidation result. Oxidation is catalyzed by the endogenous katG enzyme, which, possessing catalase-peroxidase activity, transforms isoniazid to the high reactive compounds which acetylates the specific enzymatic system presented only in Mycobacterium tuberculosis.

Thus, in result of isoniazid and catalase-peroxidase interaction, isonicotinaldehyde, isonicotinic acid and isonicotinamide are formed, which also can be formed from isonicotinoil radical and perisonicotinic acids. The mentioned compounds act on the inhA enzyme which participates in the mycolic acids biosynthesis. Mycolic acids are mycobacterium’s cellular membrane important components and hydrophilic solutions membranous barrier.

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Mycolic acids consist of long and short ,,wings,, (the short one consists of 20-24 carbon atoms, and the long one - 50-60 carbon atoms), which are formed in result of unsaturated fatty acids reduction by NADH. Isoniazid derivatives acylating the NADH, inactivate it, and prevent mycolic acids biosynthesis. Isoniazid and its derivatives selectively inhibit the mycolic acids long chains synthesis (more than 26 carbon atoms).

SAR

 The terminal basic nitrogen atom is necessary for the activity.  Hydrazine group replacing to the 2nd or 3th positions the activity decreases.  Hydrazine group’s substitution by carbonyl group: hydroxamic acid amide formation, inactive derivatives are formed.  Isoniazid hydrazones show activity, but they are unstable in the GIT (gastro-intestinal tract) and easily split forming isoniazid, which also shows activity. According to this, it has been assumed that the isoniazid derivatives activity is conditioned by isoniazid formation.

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 Inserting alkyl or аryl alkyl substituents to the isoniazid’s hydrazine group, new derivatives are formed, some of which are active, and others – no.  Inserting an alkyl substituent into the N2-position, active derivatives are obtained, but isoniazid N1-alkyl substituted derivatives’ activity decreases.

 It is possible to conclude, that no mentioned change leads to the more active derivative creation, than isoniazid.  Methazide was obtained by isoniazid molecule doubling, it form two isoniazid molecules in the body. Such doubling purpose is to slow bacterial resistance development toward the preparation.

Methazide

Metabolism

Isoniazid metabolism proceeds in various metabolic pathways; however major pathway is hydrazine primary amino group acetylation by acetyl isoniazid formation. It is necessary to mention, that some people are fast acetylators (they have N-acetyl transferase enzyme high content in the liver), and in some people the enzyme activity is very low (slow аcetylators), due to which the metabolism proceeds very slowly and drug’s undesirable effects can be expressed.

Isoniazid metabolites also are isonicotinic acid and hydrazine, acetyl hydrazine, diacetyl hydrazine, etc. Besides, isoniazid’s some quantity is excreted from the body in the glucuronides form. Isoniazid almost all metabolites are inactive.

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Isonicotinic acid thioamide derivatives

These preparations possess less activity than isoniazid, however they are active when bacteria becomes resistant to isoniazid derivatives. As these preparations are pyridoxine antagonists they can cause peripheral neuritis, and it is recommended to use pyridoxine simultaneously with ethionamide.

Action mechanism

Ethionamide action mechanism is explained similarly to isoniazid. Thus, being a prodrug, it is oxidized by catalase-peroxidase enzyme in the body transforming to the active аcylating compound – ethionamide sulfoxide. The mentioned compound inactivates inhibitor A enoyl reductase enzyme, acetylating the cysteine in the 243 position in its structure.

- S O S + NH2 NH2 C C

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N C2H5 N C2H5

SAR

Various changes were carried out in ethionamide molecule, and among them - pyridine ring substitution by benzene or pyrazine ring; thioamide substitution by аmide or thiourea, and also thioamide group replacement to the 2nd or 3th positions. All the mentioned changes lead to derivatives with weaker activity, than ethionamide.

Меtabolism

These preparations are quickly adsorbed in the body. Metabolism basically takes place in the liver and the active metabolite-sulfoxide is formed. Ethionamide’s small amount transforms to the inactive metabolites, which are 2-ethylisonicotinamid and methylated derivatives.

-O S + NH2 C

O NH2 O NH C 2 N C2H5 C

ETHIONAMIDE

N C2H5 N C H O 2 5 CH3 -O S + S NH2 NH2 C C

O N C H 2 5 O N C2H5

CH3 CH3

Nitrofuran derivates

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NItrofuran derivatives are nitrofural - furacilline, furazidin - furagin, nitrofurantoin - furadonin, furazolidone, nifurtimox.

Nitrofuran derivatives were discovered by Stillman in 1943, synthesized by Hayes in 1952 and studied by Mintzer in1953.

Nitrofuran derivatives possess action wide spectrum; they are active toward both G+ and G- bacteria and to those bacteria which are resistant to sulfonamides and antibiotics. These compounds are mainly used for urogenital and GIT infections treatment. Furadonin basically is used for cystitis, pyelonephritis, ureitis treatment. This group’s derivatives also possess antifungal, antiprotozoal- antitripanosomal activities. Furacilline is used for burns and skin infections diseases in the 0.02% aqueous, 0.066% alcohol and 0.2% ointment form. Furazidine is used for wounds and burns washings, in 1:130000 dilutions for eye drops.

They possess a lot of side effects: GIT impairment, hypersensitivity, allergic reactions. These preparations can’t be used with alcohol, because they inhibit alcohol-dehydrogenase enzyme. Thus, furazolidone is used for alcoholism treatment, when other drugs are useless or contraindicated for some reasons.

Action mechanism

Nitrofuran derivates inhibit enzymes activity, which turn piruvate to acetylCoA. This activity is due to nitro group presence, which is reduced to NHOH or NH2 in the body. Then nitrofuran reduced derivatives inhibit DNA functions and bacteria cromosoms.

According to Docampo studies nitrofuran derivates antitrepanosomal (trepanocide) activity is due to superoxide and OH radicals formation. Thus, in the first stage 5-nitrofuran derivatives form nitro radical by the nitroreductase enzyme with NADPH, which then reacts with O2 forming superoxide anion. That anion spontaneous or under the superoxide dismutase enzyme turns to H2O2 which turns to OH radical. Free radicals are very dangerous; they interact with cellular components and promote cell impairment. Treponema is highly sensitive to the free radicals, as it doesn’t contain catalase- glutathione reductase enzymatic system.

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SAR

 Replacing 5th position’s nitro group to the 3rd or 4th the activity decreases.  Second nitro groups insertion to the furan ring the activity decreases.  Substituent’s presence in the 2nd position is necessary for high antimicrobial activity.

R NO2 O

 Antimicrobial action specter is conditioned by 2nd position’s substituent nature.  In the oxazolidone ring presence (furazolidone) preparation is basically active towards to the G - bacteria, and also it is effective against lyablias and trichomonads.

Furazolidone

 In case of the amino hydantoin ring (furadonin, furagin) preparations are basically active in the urinary infections.

Furadoninum Furaginum

 Furacilline is active towards both G+ and G– bacteria.

Furacilline

Metabolism

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Nitrofurane derivates metabolism takes place in the liver and tissues. It proceeds by the nitro group reduction with active metabolite formation.

Nitroimidazole derivatives

Metronidazole Tinidazole

Metronidazole (Flagyl, Metryl), Tinidazole (Fasigyn) and Ornidazole are widely used as antimicrobial/antiprotozoal agents:

Metronidazole is a prodrug; it requires reductive activation of the nitro group by susceptible organisms. Its selective toxicity toward anaerobic and microaerophilic pathogens such as the amitochondriate protozoa T. vaginalis, E. histolytica, and G. lamblia and various anaerobic bacteria derives from their energy metabolism, which differs from that of aerobic cells These organisms, unlike their aerobic counterparts, contain electron transport components such as ferredoxins, small Fe-S proteins that have a sufficiently negative redox potential to donate electrons to metronidazole. The single electron transfer forms a highly reactive nitro radical anion that kills susceptible organisms by radical-mediated mechanisms that target DNA and possibly other vital biomolecules. Metronidazole is catalytically recycled; loss of the active metabolite's electron regenerates the parent compound. Increasing levels of O2 inhibit metronidazole-induced cytotoxicity because O2 competes with metronidazole for electrons generated by energy metabolism. Thus, O2 can both decrease reductive activation of metronidazole and increase recycling of the activated drug. Anaerobic or microaerophilic organisms susceptible to metronidazole derive energy from the oxidative fermentation of ketoacids such as pyruvate. Pyruvate decarboxylation, catalyzed by pyruvate:ferredoxin oxidoreductase (PFOR), produces electrons that reduce ferredoxin, which, in turn, catalytically donates its electrons to biological electron acceptors or to metronidazole.

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ANTIVIRAL DRUGS

Antiviral drugs are useful in tackling viral diseases where there is a lack of an effective vaccine, or where infection has already taken place. The life cycle of a virion means that for most of its time in the body it is within a host cell and is effectively disguised both from the immune system and from circulating drugs. Since it also uses the host cell's own biochemical mechanisms to multiply, the number of potential drug targets that are unique to the virus is more limited than those that can be identified for invading microorganisms. Thus, the search for effective antiviral drugs has proved more challenging than that for antibacterial drugs. Indeed, the first antiviral agents appeared relatively late on in the 1960s, and only three clinically useful antiviral drugs were in use during the early 1980s.

Antiviral research has been aided by advances in viral genomics and genetic engineering, as well as the use of X-ray crystallography and molecular modeling. Although the genetic sequence is unlikely to be identical from one virus to another, it is possible to identify similar genes coding for similar proteins with similar functions. These proteins can then be studied as potential drug targets.

Good drug targets are proteins which are likely to have the following characteristics:

• They are important to the life cycle of the virus, such that their inhibition or disruption has a major effect on infection.

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• They bear little resemblance to human proteins, thus increasing the chances of good selectivity and minimal side effects.

• They are common to a variety of different viruses and have a specific region which is identical in its amino acid composition. This makes the chances of developing a drug with broad antiviral activity more likely.

• They are important to the early stages of the virus life cycle, so that the virus has less chance of spreading through the body and producing symptoms.

Classification

1. Antiviral drugs used against DNA viruses (herpes viruses)

 Inhibitors of viral DNA polymerase 2. Antiviral drugs acting against RNA viruses: flu virus

 Ion channel disrupters: adamantane derivatives  Neuraminidase inhibitors 3. Antiviral drugs acting against RNA viruses: HIV

 Nucleoside reverse transcriptase inhibitors  Non-nucleoside reverse transcriptase inhibitors  HIV protease inhibitors 4. Antiviral drugs acting against RNA viruses: cold virus

5. Broad-spectrum antiviral drugs

Antiviral drugs used against DNA viruses

Most of the drugs which are active against DNA viruses have been developed against herpes viruses to combat diseases such as cold sores, genital herpes, chickenpox, shingles, eye diseases, viral lymphoma, and Kaposi's sarcoma. Nucleoside analogues have been particularly effective. Aciclovir was discovered by compound screening and was introduced into the market in 1981. It represented a revolution in the treatment of herpes infections, being the first relatively safe, non-toxic drug to be used systemically.

Aciclovir has a nucleoside-like structure and contains the same nucleic acid base as deoxyguanosine, but lacks the complete sugar ring.

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In virally infected cells, it is phosphorylated by the viral thymidine kinase and cellular thymidylate kinase enzymes in three stages to form a triphosphate which is the active agent, and so aciclovir itself is a prodrug.

Nucleotide triphosphates are the building blocks for DNA replication where a new DNA strand is constructed using a DNA template-a process catalyzed by the enzyme DNA polymerase. Aciclovir triphosphate prevents DNA replication in two ways. First, it is sufficiently similar to the normal deoxyguanosine triphosphate building block that it can bind to DNA polymerase and inhibit it. Second, DNA polymerase can catalyze the attachment of the aciclovir nucleotide to the growing DNA chain. Since the sugar unit is incomplete and lacks the required hydroxyl group normally present at position 3' of the sugar ring, the nucleic acid chain cannot be extended any further. Thus, the drug acts as a chain terminator.

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However, what is to stop aciclovir triphosphate inhibiting DNA polymerase in normal, uninfected cells? The answer lies in the fact that aciclovir is only converted to the active triphosphate in infected cells. The explanation for this lies in the first phosphorylation reaction catalyzed by the enzyme thymidine kinase. Although this enzyme is present in host cells, the herpes virus carries its own version. It turns out that aciclovir is more readily converted to its monophosphate by viral thymidine kinase (100-fold) than by host cell thymidine kinase. Once formed, the monophosphate is converted to the active triphosphate by cellular enzymes. In normal uninfected cells, therefore, aciclovir is a poor substrate for cellular thymidine kinase and remains as the prodrug. This, along with the fact that there is a selective uptake of aciclovir by infected cells, explains its excellent activity and much reduced toxicity relative to previous drugs. Another feature which enhances its safety is that aciclovir triphosphate shows a 50-fold selective action against viral DNA polymerases relative to cellular polymerases.

Aciclovir has disadvantages:

1. The oral bioavailability of aciclovir is quite low and to overcome this, various prodrugs were developed to increase lipophilicity. Valaciclovir is an L-valylester prodrug and is hydrolysed to aciclovir in the liver and gut wall. Desciclovir is an analogue of aciclovir which lacks the carbonyl group at position 6 of the purine ring. Once in the blood supply, metabolism by cellular xanthine oxidase oxidizes the 6-position to give aciclovir.

2. Unfortunately, strains of herpes are appearing which are resistant to aciclovir. This can arise due to mutations, either of the viral thymidine kinase enzyme such that it no longer phosphorylates aciclovir, or of viral DNA polymerase such that it no longer recognizes the activated drug. Aciclovir is not effective against all types of herpes virus. There are eight herpes viruses which are divided into 153 three subfamilies. Aciclovir is effective against the α-subfamily but not the β-subfamily, because the latter produces a different thymidine kinase that fails to phosphorylate acyclovir. The analogue gancidovir, however, having more similarity to the natural substrate, is phosphorylated by thymidine kinases produced by both the α- and β-subfamilies and can be used against both viruses. Ganciclovir contains an extra hydroxymethylene group which increases its similarity to deoxyguanosine.

Unfortunately, the drug is not as safe as aciclovir as it can be incorporated into cellular DNA. Nevertheless, it can be used for the treatment of cytomegalovirus (CMV) infections. Since ganciclovir has a low oral bioavailability, the valine prodrug - valganciclovir was created.

Some viruses are immune from the action of the above antiviral agents because they lack the enzyme thymidine kinase. As a result, phosphorylation fails to take place. Cidofovir was designed to combat this problem. It is an analogue of deoxycytidine 5-monophosphate where the sugar and phosphate groups have been replaced by an acyclic group and a phosphonomethylene group respectively. The latter group acts as a bioisostere for the phosphate group and is used because the phosphate group itself would be more susceptible to enzymatic hydrolysis.

Since a phosphate equivalent is already present, the drug does not require thymidine kinase to become activated. Cidofovir is a broad-spectrum antiviral agent which shows selectivity for viral DNA polymerase. Unfortunately, the drug is extremely polar and has a poor oral bioavailability and it is also toxic to the kidneys.

KEY POINTS

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• Nucleoside analogues have been effective antiviral agents used against DNA viruses, mainly herpes viruses.

• Nucleoside analogues are prodrugs which require to be phosphorylated to a triphosphate in order to be active.

• They have a dual mechanism of action whereby they inhibit viral DNA polymerase and also act as DNA chain terminators.

• Nucleoside analogues show selectivity for virally infected cells over normal cells if viral thymidine kinase is required to catalyse the first of three phosphorylation steps.

• They are also taken up more effectively into virally infected cells and their triphosphates inhibit viral DNA polymerases more effectively than cellular DNA polymerases.

• Agents containing a bioisostere for a phosphate group can be used against DNA viruses lacking thymidine kinase.

Antiviral drugs acting against RNA viruses: flu virus

Neuraminidase inhibitors. The flu virus contains (-) ssRNA and has two glycoproteins called HA and neuraminidase in its outer membrane. In order to reach the epithelial host cells of the upper respiratory tract the virus has to negotiate a layer of protective mucus, and it is thought that the viral protein NA is instrumental in achieving this. The mucosal secretions are rich in glycoproteins and glycolipids which bear a terminal sugar substituent called sialic acid (also called N-acetylneuraminic acid). Neuraminidase (also called sialidase) is an enzyme which is capable of cleaving the sialic acid sugar moiety from these glycoproteins and glycolipids, thus degrading the mucus layer and allowing the virus to reach the surface of epithelial cells. Once the virus reaches the epithelial cell, adsorption takes place whereby the virus binds to cellular glycoconjugates which are present in the host cell membrane, and which have a terminal sialic acid moiety. The viral protein HA /hemagglutinin/ is crucial to this process. Like NA, it recognizes sialic acid but instead of catalyzing the cleavage of the sialic acid from the glycoconjugate, HA binds to it. Once the virus has been adsorbed, the cell membrane bulges inwards taking the virus with it to form a vesicle called an endosome-a process is called receptor-mediated endocytosis. The pH in the endosome then decreases, causing HA in the virus envelope to undergo a dramatic conformational change whereby the hydrophobic ends of the protein spring outward and extend towards the endosomal membrane. After contact, fusion occurs and the RNA nucleocapsid is released into the cytoplasm of the host cell.

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Since NA plays two crucial roles in the infectious process, it is a promising target for antiviral drugs creation against flu virus. Neuraminidase is a mushroom-shaped tetrameric glycoprotein anchored to the viral membrane by a single hydrophobic sequence of some 29 amino acids. As a result, the enzyme can be split enzymatically from the surface and studied without less of antigenic or enzymic activity. X-ray crystallographic studies have shown that the active site is a deep pocket located centrally on each protein subunit. There are two main types of the enzyme (corresponding to the influenza viruses A and B) and various subtypes. However, the 18 amino acids making up the active site itself are constant. This gives chance to design inhibitor’s molecule, which will show selective antiviral activity.

The most important interactions of the sialic acid with enzyme are:

 Involved the carboxylate ion of sialic acid in ionic interactions and hydrogen bonds with three arginine residues, particularly with Arg-371. In order to achieve these interactions, the sialic acid has to be distorted from its most stable chair conformation (where the carboxylate ion is in the axial position) to a pseudoboat conformation where the carboxylate ion is equatorial.  The glycerol side chain of sialic acid fills one of these pockets, interacting with glutamate residues and a water molecule by hydrogen bonding.  The hydroxyl group at C-4 of sialic acid is situated in another binding pocket interacting with a glutamate residue.  Finally, the acetamido substituent of sialic acid fits into a hydrophobic pocket which is important for molecular recognition. This pocket includes the hydrophobic residues Trp and Ile which lie close to the methyl carbon (C-11) of sialic acid as well as the hydrocarbon backbone of the glycerol side chain.  It was further established that the distorted pyranose ring binds to the floor of the active site cavity through its hydrophobic face. The glycosidic OH at C-2 is also shifted from its normal equatorial position to an axial position where it points out of the active site and can form a hydrogen bond to Asp-l51, as well as an intramolecular hydrogen bond to the hydroxyl group at C-7. Based on these results, a mechanism of hydrolysis was proposed which consists of four major steps.

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1. The first step involves the binding of the substrate (sialoside) as described above.

2. The second step involves proton donation from the activated water facilitated by the negatively charged Asp, and formation of an endocyclic sialosyl cation transition state intermediate. Glu is proposed to stabilize the developing positive charge on the glycosidic oxygen as the mechanism proceeds.

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3. Support for the proposed mechanism comes from kinetic isotope studies which indicate it is an SN1 nucleophilic substitution. This is consistent with an SN1 mechanism having a high degree of stereo facial selectivity.

4. The final step of the mechanism is release of sialic acid. NMR studies have also been carried out which indicate that sialic acid is released as the α-anomer. Possibly expulsion of the product from the active site is favoured by mutarotation to the more stable β-anomer.

Finally, site directed mutagenesis studies have shown that the activity of the enzyme is lost if Arg is replaced by lysine and Glu by aspartate. These replacement amino acids contain similarly charged residues but have a shorter residue chain. As a result, the charged residues are unable to reach the required area of space in order to stabilize the intermediate.

Transition-state inhibitors

The transition state shown has a planar trigonal centre at C-2 and so sialic acid analogues containing a double bond between positions C-2 and C-3 were synthesized to achieve that same trigonal geometry at C-2.

This resulted in the discovery of the inhibitor 2-desoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en). In order to achieve the required double bond, the hydroxyl group originally present at C-2 of sialic acid had to be omitted, which resulted in lower hydrogen bonding interactions with the active site. On the other hand, the inhibitor does not need to distort from a favourable chair shape in order to bind, and the energy saved by this more than compensates for the loss of the one hydrogen bonding interaction. Unfortunately, this compound also inhibited bacterial and mammalian sialidases and could not be used therapeutically.

The most important result from further studies was the discovery that the region around the 4-OH of sialic acid could interact with an ammonium or guanidinium ion. As a result, sialic acid analogues, having an amino or guanidinyl group at C-4 instead of hydroxyl, were modeled in the active site to study the binding interactions and to check whether there was room for the groups to fit.

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Molecular modeling studies had suggested that the larger guanidinium group would be capable of even greater hydrogen bonding interactions as well as favorable van der Waals interactions. The relevant structure was indeed found to be a more potent inhibitor having a 100-fold increase in activity. Zanamivir is a slow-binding inhibitor with a high binding affinity to influenza A neuraminidase. Unfortunately, the polar nature of the molecule means it has poor oral bioavailability and it is administered by inhalation.

Following on from the success of these studies, 4-epi-amino-Neu5Ac2en was synthesized. This structure proved to be a better inhibitor than Neu5Ac2en but not as good as zanamivir. The pocket into which this amino group fits is small and there is no room for larger groups.

A problem with the inhibitors described above is their polar nature. The glycerol side chain is particularly polar and has important binding interactions with the active site. However, it was found that it could be replaced by a carboxamide side chain with retention of activity. A series of 6- carboxamide analogues was prepared to explore their structure activity relationships.

 Secondary carboxamides where Rcis = H showed similar weak inhibition against both A and B forms of the neuraminidase enzyme.  Tertiary amides having an alkyl substituent at the cis position resulted in a pronounced improvement against the A form of the enzyme, with relatively little effect on the activity against the B form.

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Thus, when carboxamide group is inserted, in contrast to glycerol residue it can’t interact with glutamic acid residue, in this result it changes its torsion angle forming salt bridge with the arginine residue, which, however, leads to the A type virus enzyme conformation little change. In contrast to this, binding with type B enzyme, conformation significant change was observed due to formed bridge. This is the reason, that higher energy is required for carboxamides binding with type B enzyme, and, therefore, selectivity toward type A enzyme is more.  Good activity was related to a variety of different-sized Rtrans substituents larger than methyl, but the size of the Rcis group was more restricted and optimum activity was achieved when Rcis was ethyl or n-propyl.

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 The 4-guanidino analogues are more active than corresponding 4-amino analogues but the improvement is slightly less than that observed for the glycerol derivatives, especially where the 4-amino analogue is already highly active. The dihydropyran oxygen of Neu5Ac2en and related inhibitors has no important role to play in binding these structures to the active site of neuraminidase. Therefore, it should be possible to replace it with a methylene isostere to form carbocyclic analogues.

This would have the advantage of removing a polar oxygen atom which would increase lipophilicity and potentially increase oral bioavailability. Moreover, it would be possible to synthesize cyclohexene analogues such as structure II which more closely match the stereochemistry of the reaction's transition state than previous inhibitors compare the reaction intermediate which can be viewed as a transition-state mimic. Such agents might be expected to bind more strongly and be more potent inhibitors. Structures I and II were synthesized to test this theory, and it was discovered that structure II was 40 times more potent than structure I as an inhibitor. Both structures have half chair conformations, but these are different due to the position of the double bond. It was now planned to replace the hydroxyl group on the ring with an amino group to improve binding interactions and to remove the glycerol side chain to reduce polarity.

A series of alkoxy analogues of this structure have synthesized in order to maximize hydrophobic interactions. The most potent of the above analogues was the pentyloxy derivative. Oseltamivir (Tamiflu) is the ethyl ester prodrug of thus compound. It is taken orally and is converted to active drug by esterases in the gastrointestinal tract.

Antiviral drugs acting against RNA viruses: HIV

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Until 1984, no anti-HIV drug was available, but an understanding of the life cycle of HIV has led to the identification of several possible drug targets. It is known that HIV

 is an example of a group of viruses known as the retroviruses.  There are two variants of HIV: HIV-1 and HIV-2.  Contains two identical strands of (+) ssRNA within its capsid.  Also present are the 3 viral enzymes (reverse transcriptase, integrase and protease), as well as other proteins called p7 and p9.  The capsid is made up of protein, and surrounding the capsid there is a layer of matrix protein (p17),  Then a membranous envelope which originates from host cells and which contains the viral glycoproteins gp120 and gp41. Both of these proteins are crucial to the processes of adsorption and penetration.  Disintegration of the protein capsid then takes place, probably aided by the action of a viral enzyme called protease. Viral RNA and viral enzymes are then released into the cell cytoplasm.  The conversion of RNA into DNA is not a process that occurs in human cells, so there are no host enzymes to catalyse the process. Therefore, HIV carries its own enzyme-reverse transcriptase-to do this.  Viral DNA insertion into the human DNA is catalyzed by viral integrase enzyme. Since 1987 most drugs that have been developed act against the viral enzymes reverse transcriptase and protease. However, a serious problem with the treatment of HIV is the fact that the virus undergoes mutations extremely easily. This results in rapid resistance to antiviral drugs. Experience has shown that treatment of HIV with a single drug has a short –term benefit, but in the long term the drug serves only to select mutated viruses which are resistant. As a result, current therapy involves combinations of different drugs acting on both reverse transcriptase and protease.

The available drugs for highly active antiretroviral therapy include:

 Nucleoside reverse transcriptase inhibitors /zidovudine, lamivudine, didanosine, etc./  Non-nucleoside reverse transcriptase inhibitors /nevirapine, delavirdine/  HIV protease inhibitors /saquinavir, ritonavir, indinavir, nelfinavir/. Nucleoside reverse transcriptase inhibitors

Since the enzyme reverse transcriptase is unique to HIV, it serves as an ideal drug target. Nevertheless, the enzyme is still a DNA polymerase and care has to be taken that inhibitors do not

162 have a significant inhibitory effect on cellular DNA polymerases. Various nucleoside-like structures have proved useful as antiviral agents. The vast majority of these are not active themselves but are phosphorylated by three cellular enzymes to form an active nucleotide triphosphate. This is the same process previously described above, but one important difference is the requirement for all three phosphorylations to be carried out by cellular enzymes as HIV does not produce a viral kinase.

Zidovudine is an analogue of deoxythymidine, where the sugar 3'-hydroxyl group has been replaced by an azido group. It inhibits reverse transcriptase as the triphosphate. Furthermore, the triphosphate is attached to the growing DNA chain. Since the sugar unit has an azide substituent at the 3' position of the sugar ring, the nucleic acid chain cannot be extended any further. Unfortunately, zidovudine can cause severe side effects such as anemia.

Studies of the target enzyme have allowed the development of less toxic nucleoside analogues such as lamivudine (an analogue of deoxycytidine where the 3' carbon has been replaced by sulfur). This drug has also been approved for treatment of hepatitis B. These compounds are DNA chain inhibitors.

Non-nucleoside reverse transcriptase inhibitors

The NNRTls are generally hydrophobic molecules that bind to an allosteric binding site which is hydrophobic in nature.

Since the allosteric binding site is separate from the substrate binding site, the NNRTIs are non- competitive, reversible inhibitors. X-ray crystallographic studies on enzyme-inhibitor complexes 163 show that the allosteric binding site is adjacent to the substrate binding site. Binding of an NNRTI to the allosteric site results in an induced fit which locks the neighboring substrate-binding site into an inactive conformation. NNRTls show a higher selectivity for HIV-1 reverse transcriptase over host DNA polymerases than do the NRTIs. Second- and third-generation NNRTls were developed specifically to find agents that were active against resistant variants as well as wild-type virus.

Protease inhibitors

The HIV protease enzyme is a dimer made up of two identical protein units, each consisting of 99 amino acids.

This enzyme has a number of differences from the human proteases:

 The enzyme has broad substrate specificity and can cleave a variety of peptide bonds in viral polypeptides, but crucially it can cleave bonds between a proline residue and an aromatic residue (phenylalanine or tyrosine). The cleavage of a peptide bond next to proline is unusual and does not occur with mammalian proteases such as renin.  The viral enzyme and its active site are symmetrical. This is not the case with mammalian proteases, again suggesting the possibility of drug selectivity.  There are eight binding subsites in the enzyme, four on each protein unit, located on either side of the catalytic region and are numbered Sl-S4 on one side and Sl'-S4' on the other side. The relevant residues on the substrate are numbered P1-P4 and P1'-P4'. As peptides bond splitting occurs by transition-state intermediate compound formation, the structure was based on for the inhibitor modeling and various isostere substitutions were carried out in the molecule to increase intermediate compound stability.

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Transition-state inhibitors are designed to mimic the transition state of an enzyme catalyzed reaction. The design of saquinavir started by considering a viral polypeptide substrate and identifying a region of the polypeptide which contains a phenylalanine-proline peptide link. The peptide link normally hydrolysed is between Phe-167 and Pro-168, and so this link was replaced by a hydroxyethylamine transition-state isostere to give a structure which successfully inhibited the enzyme. The peptide link was replaced by the hydroxylamine transition-state isostere and the resulting N- and C-protected structure was tested and found to have weak inhibitory activity. The inclusion of an asparagine group (structure II) to occupy the S2 subsite resulted in a 40-fold increase in activity. An X-ray crystallographic study of the enzyme-inhibitor complex was carried out and revealed that the protecting group (Z) occupied the S3 subsite, which proved to be a large hydrophobic pocket. As a result, it was possible to replace this protecting group with a larger quinoline ring system to occupy the subsite more fully and lead to a six-fold increase in activity (structure III). For additional interactions formation decahydroisoqinoline and isobutyl hydrophobic groups were inserted. The resulting structure was saquinavir having a further 60-fold increase in activity.

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Broad-spectrum antiviral drugs. Interferons.

Interferons are natural proteins, which are potent cytokines and possess antiviral, immune modulator and antiproliferative properties. They are synthesized in the host infected cells by different signals influence, especially viruses and inhibit their replication and proteins synthesis; otherwise, they block viral cells providing intracellular immune response. These compounds are used for flu, herpes, hepatitis and cold treatment. There are human interferons 3 subgroups, depending on their production source:

 α-interferons (alferon)- are synthesized by lymphocytes and macrophages and represent most used interferons subgroup;  β-interferons (betaseron)- are produced by the fibroblasts and epithelial cells;  γ-interferons - are produced in the T-and killer cells by antigenic signals, mitogens and specific cytokines. It has been discovered only one subtype of these interferons in contrast to the α, β- interferons. γ-interferons have weaker antiviral activity than α, β-interferons and basically possess immune regulating effect. Interferons can’t be used per oral due to low bioavailability (not absorbed in the GIT) are used in i/m or s/c injections form. Pharmacological effect is quite long: approximately 6 days.

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DRUGS ACTING ON ENDOCRINE SYSTEM

In 1902, Baylis and Starling demonstrated that the effect exerted by the duodenum on pancreatic secretion was due to a blood borne factor that seemingly acted as a “chemical messenger.” Their research led to the identification of this chemical messenger, which they called secretin. They went on to suggest that secretin was not unique, hypothesizing that many chemical agents are secreted by various cells throughout the body and that these agents, upon distribution by the bloodstream and circulation, influence the function of organs that are located some distance away. They coined the term hormone to describe such chemical messengers that are synthesized in one organ system and distributed via the circulation to distant organ systems to elicit an altered biochemical response.

Hormones are central to homeostasis since they facilitate chemical control over metabolic and biochemical processes throughout the entire body. The endocrine system, which exerts control over chemical processes via hormones, is crucial to homeostasis over an intermediate time scale. The endocrine system is distinct from the nervous system, which employs neurotransmitters to control electrical and electrochemical processes and to influence homeostasis over a shorter time scale. The endocrine system is also distinct from the immune system, which employs immunomodulators to control cellular processes and to influence homeostasis over a longer-term time scale.

The endocrine system is composed of hormone-producing organs within the body, starting from nervous system – hypophysis, hypothalamus, till various glands, as thyroid gland (thyroxine), pancreas (insulin) and other organs – kidneys (renin) etc.

Hormones are ideal targets for rational drug design. Since hormones are precise chemical messengers influencing specific metabolic function throughout the body, their pharmacological manipulation, by the administration of either agonists or antagonists, permits therapeutic modulation of a wide range of biochemical events. Moreover, since many hormones are small molecules (i.e., steroids or short peptides with molecular weights less than 1000) they are readily studied using molecular modeling calculations to facilitate rational drug design.

There are different classifications for hormones, but the most convenient in medicinal chemistry is the classification according to chemical structure: steroid hormones, peptide hormones and amines.

Steroid hormones 167

The steroid hormones include the sex hormones (estrogens, progestins, androgens) and the adrenocorticoids (glucocorticoids, mineralocorticoids).

The adrenocorticoids and sex hormones have much in common. They all are steroids; consequently, the rules that define their structures, chemistry, and nomenclature are the same. The rings of these biochemically dynamic and physiologically active compounds have a similar stereochemical relationship. Changes in the geometry of the ring junctures generally result in inactive compounds regardless of the biological category of the steroid. Similar chemical groups are used to render some of these agents’ water soluble or active when taken orally or to modify their absorption.

In addition, the adrenocorticoids and the sex hormones, which include the estrogens, progestins, andandrogens, are biosynthesized mainly from cholesterol.

Cholesterol structure

Moreover, many of the metabolic reactions are similar for these compounds. For example, reduction of double bonds at positions 4-5 or 5-6, epimerization of 3-hydroxyl groups, reduction of 3-keto groups to the 3-hydroxyl groups and oxidative removal of side chains are transformations common to these agents.

Despite the similarities in chemical structures and stereochemistry, each class of steroids demonstrates unique and distinctively different biological activities. Minor structural modifications to the steroid nucleus, such as changes in or insertion of functional groups at different positions, cause marked changes in physiologic activity.

Steroid hormones nomenclature and structure

Steroids consist of four fused rings (A, B, C, and D). Chemically, these hydrocarbons are cyclopentanoperhydrophenanthrenes; they contain a five-membered cyclopentane (D) ring plus the three rings of phenanthrene. A perhydrophenanthrene (rings A, B, and C) is the completely saturated derivative of phenanthrene (fig. 1). The polycyclic hydrocarbon known as 5α-cholestane will be used to illustrate the numbering system for a steroid. The term “cholestane” refers to a steroid with 27 carbons that includes a side chain of eight carbons at position 17. Numbering begins in ring A. The angular methyl groups are numbered 18 (attached to C13) and 19 (attached to C10). The 17 side chain begins with C20; the number of carbon atoms in this side chain can vary depeding on steroid type.

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Fig. 1. Steroid hormones structure, nomenclature

Using the planar representation for drawing the steroid structure, the cyclopentanoperhydrophenanthrene structure becomes a plane with two surfaces: A top or β-surface is pointing out toward the reader, and the bottom or α-surface is pointing away from the reader. Hydrogens or functional groups on the β-side of the molecule are denoted by solid lines; those on the α-side are designated by dotted lines.

Fig. 2. Orientation of steroid ring substituents

If the opposite substituents (5-10, 8-9, 13-14) in the parts of rings juncture have /β or β/ orientation, then the rings have trans configuration. E.g. in 5-cholestane the hydrogen atom at C5 has , and the C19 angular methyl group has β orientation making the A/B ring juncture trans. Similarly, the configuration of the 8 and 9β hydrogens, and the 14 hydrogen and C18 angular methyl group gives trans configuration to the rings B/C and C/D. In steroids structure B/C and C/D rings always are in trans configuration, whereas A/B rings can also have cis conformation (fig. 3). Side chain in C17 position always has β orientation.

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Fig. 3. Conformations of steroids’ saturated rings

It is known that six-membered cyclohexane ring can exist in two forms: chair and boat, besides, the substituents can have axial (a) or equatorial (e) position. In 5-cholestane structure, as well as in steroid hormones structure, six-membered rings have boat and five-membered rings – chair conformation (fig. 4).

β a

β e α e

α a

a) b)

Fig. 4. a) plane and conformational structure of 5-cholestane;

b) axial (a) and equatorial (e) orientation of substituents

Although cyclohexane may undergo to boat conformation, steroids are rigid structures, because they generally have at least one transfused ring system and these rings must have only chair conformation.

In most of the important steroids, a double bond is present between positions 4-5 or 5-6; consequently, there is no cis or trans relationship between rings A/B. The symbol ∆ often is used to designate a carbon–carbon double bond (C=C) in a steroid. If the C=C is between positions 4-5, the compound is referred to as a ∆4-steroid. If the C=C is between positions 5-6, the compound is designated a ∆5-steroid, but if between 5-10, the compound is designated a ∆5(10)-steroid. E.g. cholesterol (cholest-5-en-3-ol) is a ∆5-steroid or, more specifically, a ∆5-sterol, because it is an unsaturated alcohol. In case of double bond insertion ring A changes its conformation (fig. 5) from complete chair (A) to half-chair (B); in case of two double bonds insertion - to plane boat (C).

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Fig. 5 Possible conformations of ring A

Rings stereochemistry has a significant influence on biological activity of these group substances. All these compounds have cholestane-type conformation.

Biologically active compounds include members of the 5-pregnane, 5-androstane, and 5- estrane steroid classes (fig. 6).

Pregnanes are steroids with 21 carbon atoms. From adrenocorticoids (adrenal cortex hormones) cortisone and hydrocortisone, and progesterone from female sex hormones are pregnane analogues.

Fig. 6. Steroid hormones classification according to chemical structure 171

The male sex hormones androgens (testosterone) are based on the structure of androstane containing 19 carbon atoms.

Estranes are steroids with 18 carbon atoms, with the C19 angular methyl group at C10 replaced by hydrogen. These hormones are female sex hormones - estradiol and contain aromatic ring A.

Steroid hormones action mechanism

In addition to their structural similarities, one of the specificites of steroid hormones is their receptors. Unlike other hormones, mediators and drugs which receptors are proteins that are embedded in the cell membranes, final targets of steroids are the genes located in the nucleus. Steroid receptors are highly specific macromolecules found in central regulatory organs, e.g., hypothalamus, pituitary (hypophysis), in various end-point target tissues, e.g., uterus, vagina, prostate, and in lower concentrations in the brain, liver, kidney, ovary, and many other organs. Steroid hormones exhibit remarkable tissue selectivity when binding to these receptors. Within the target organs in which the steroids bind, the steroid molecules exert their influence directly on protein synthesis, at the level of transcription of the genetic message. Steroid hormones work primarily by regulating tissue-specific gene expression; the hormones enter the nucleus of a cell, bind to target genes in the DNA of that cell, and subsequently influence protein synthesis. Although steroids may also affect other cellular processes by influencing various enzyme systems through cAMP dependent protein kinases, their effects on protein synthesis are of primary importance.

Thus, taking into consideration the general regularities of steroids’ structure, the adrenocorticoids, estrogenes, progestines and androgens share a common mode of action. While the macromolecules and target tissues involved show extreme specificity for the appropriate steroid hormones and their congeners, the general scheme of the steroid–receptor mechanism is remarkably uniform.

Fig. 7. Steroid hormone receptors

Steroids are present in the body only in extremely low concentrations (e.g., 0.1–1.0 nM), but they exert potent physiologic effects on sensitive tissues, and they bind with high affinity to intracellular receptors. As was mentiones before, the steroid hormones act on target cells to regulate gene expression and protein biosynthesis via binding to the receptors and formation of steroid–receptor complexes. 172

The general steroid–receptor hypothesis is based mainly on estrogen and progesterone receptors. The currently accepted mechanism is unique and consists of several steps at different subcellular structures:

1. Cytoplasmic receptor activation 2. Translocation of the hormone–receptor complex to the nucleus 3. Binding of the complex to DNA acceptor sites 4. Activation of transcription and influencing protein synthesis The lipophilic steroid hormones are transported to their target cells via the blood stream in a protein-bound form (reversible binding), but diffuse into the cell as free steroids. At this point, they encounter a cytoplasmic steroid receptor protein. These receptors are large proteins; the estrogen receptor, for example, has a molecular weight of approximately 75,000. These receptors have two functions: they bind to the steroids and act as transcriptional factors via interaction with specific DNA site. Early studies suggested that the unoccupied steroid receptors were located solely in the cytosol of target cells. Recent investigations on estrogen, progestin, and androgen action, however, indicate that active, unoccupied receptors also are present in the nucleus of the cell.

The general model of the steroid receptor protein consists of several functional domains:

Fig. 8. Steroid receptors’ domains

- “E” domain (ligand binding region) is composed of the C-terminal 250 amino acids; this section has the steroid binding site and additional sites for binding to chaperone proteins. - “D” domain (hinge region), located adjacent to the “E” domain, is involved with the translocation of the steroid–receptor complex into the nucleus of the target cell. - “C” domain (DNA binding region) is made up of 70 amino acids clumped into two finger-shaped regions, each coordinated with a Zn ion; these so-called zinc fingers are crucial to the process whereby the steroid recognizes and binds to the DNA once the nucleus has been penetrated. - finally, at the N-terminal of the steroid receptor protein is the “A/B” domain (DNA modulator region), which enables the steroid–receptor complex that has bound to the DNA to activate genes and initiate transcription. As was mentioned before “E” domain is bound to chaperone peptides and consist of macromolecules such as heat shock proteins (e.g., hsp70, hsp90). Hsp90 heat shock protein increases the receptor affinity towards the hormone and inhibits its affinity towards the cell nucleus components. The chaperone peptides help to twist and turn the steroid receptor protein into an improved three-dimensional shape for final and optimal binding of the steroid molecule. Following binding of the chaperone peptides, the steroid-hormone–receptor complex becomes “mature steroid- 173 hormone–receptor complex”. Following optimal binding of the steroid to its receptor, the mature complex dissociates, releasing the chaperones and converting the steroid hormone receptor complex into an activated form. The activated receptor is then phosphorylated, dimerized, and transported into the nucleus with the aid of the D domain. Once in the nucleus, the zinc fingers of the C domain bind to the DNA (hormone responsive elements - HRE) and the A/B domain effects gene activation. The synthetic response in the cell is very rapid: within 15 minutes a considerable increase in the concentration of RNA polymerase can be detected, and within 30 minutes induced protein synthesis is measurable.

Additional evidence suggests that steroid receptors may activate transcription in the absence of hormone due to other mechanisms which bring to phosphorylation.

Fig. 9. Steroids action mechanisms

The steroid–receptor complex remains in the nucleus for a limited time only, and eventually dissociates from the chromatin. About 40% of the receptors released in this dissociation are recycled and used again; the rest 60% are destroyed and resynthesized. Steroid hormones can even regulate the level of synthesis of their own receptors, and sometimes the synthesis of other steroid receptors as well.

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Adrenocorticoid drugs

It is known that adrenocorticoids – mineralocorticoids and glucocorticoids are synthesized in the adrenal glands cortex. Pregnenolone, synthesized from cholesterol, is the precursor of adrenocorticoids’ synthesis. The synthesis of mineralocorticoids (which regulate the organism electrolyte balance) and glucocorticoids (which regulate carbohydrate, lipid and protein metabolism) is regulated by different mechanisms. Aldosterone synthesis is regulated by rennin-angiotensin system activation; glucocorticoids’ synthesis undergoes pituitary-hypothalamic regulation. Adrenocoticoids both classes are important for new drugs creation.

Glucocorticoids promote gluconeogenesis, lipolysis, stimulate the release of amino acids during the course of muscle catabolism, increase the rate of enzyme synthesis in the nucleus of target cells. The principal target of glucocorticoids is the liver, although other organs notably the muscles and brain are also rich in glucocorticoid receptors. Most glucocorticoids have some mineralocorticoid effect, which is usually considered an undesirable activity. Through structural chemistry and structure– activity relationship (SAR) studies, molecular modifications can separate the two activities. Glucocorticoids provide a valuable lesson in drug design. Since they influence so many enzymes in so many cell types, the pharmacological effects of glucocorticoids are likewise many and far reaching. However, so too are their side effects. If the drug designer is targeting a receptor that has widespread distribution and is not localized to a single tissue or cell type, the likelihood of unwanted side effects is concomitantly increased.

The glucocorticoid receptor belongs to a super family of nuclear receptor proteins that includes steroids. Although the glucocorticoid receptor differs from the estrogen or progesterone receptors, the basic principles of its action seem to be the same. The glucocorticoid receptor is 800 amino acids in length and has three functional domains: the C-terminal has the glucocorticoid binding region, the middle portion has the DNA binding region (containing nine cysteine residues folded into a “two finger” structure stabilized by Zn2+ ions), and the N-terminal, which has the receptor-specific region. Upon entering the cell, it binds to the glucocorticoid receptor, which is itself bound to two stabilizing proteins, including two molecules of heat shock protein (Hsp90). When the steroid binds, the complex becomes unstable and the Hsp90 molecules are released. The steroid–receptor complex is now able to enter the nucleus within the cell as an activated dimer. The complex then binds to DNA have its action.

The most important clinical application of glucocorticoids and their semisynthetic analogues is their anti-inflammatory activity, as they inhibit both the cellular (act on leucocytes) and molecular mediators (inhibit the inflammatory cytokines, chemokines and others) of inflammation. They are also effective in replacement therapy in those patients having glucocorticoids insufficiency.

Structure-activity relationship (SAR)

The structure–activity relationships of glucocorticoids are based on two natural hormones, cortisol and corticosterone.

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The characteristic structural features of these hormones are the conjugated 3-ketone, the 11-OH group, and the l7β-ketol side chain. They are ∆4-derivatives. Molecular modifications have been aimed at deriving compounds with glucocorticoid and anti-inflammatory actions but lacking in mineralocorticoid effects and other side effects. Substituents added to the molecule may alter bioactivity, e.g. replacement of 21-OH group with fluorine atom increases the sodium-retaining activity of glucocorticoid, but in case of chlorine or bromine the same activity disappears. There are derivatives lacking 3-keto group but still possessing significant effect; it means that the mentioned group have minor influence on drug specific action and steroid-receptor binding.

It is interesting to note that progesterone binds well to the glucocorticoid receptor despite a missing 11-oxygen functional group, but it fails to elicit gene activation in glucocorticoid target cells, thus shedding light on the role of the 11-OH group.

The optimum glucocorticoid structure shows a 1α, 2β-half-chair conformation for ring A. As was mentioned before absolute trans conformation for ring B/C and C/D is mandatory.

Fig. 10. Hydrocortisone and prednisolone conformations

SAR studies show that C and D rings and their substituents are more important for receptor binding than A and B rings. Generally, insertion of bulky substituentson the β-side of the molecule

176 abolishes glycogenic activity, whereas insertion on the -side does not. It has been suggested that association of these steroids with receptors involves β-surfaces of rings C and D and the17β-ketol side chain (due to hydrogen bonds). It is possible, however, that association with the -surface of rings A, C, and D, as well as their substituents 17, 16, 16β, is essential for sodium-retaining activity. Many functional groups, such as 17-OH, 16-CH3, 16β-CH3, 16-CH3O, 16-OH, abolish or reverse this activity in 11-desoxycorticosterone and 11-oxygenated steroids. Discussions of exceptions of these generalities are also found in the literature.

Cortisol (hydrocortisone) is used as a hormone-replacing and anti-inflammatory drug. To obtain necessary pharmacokinetic properties for hydrocortisone, as well as for other glucocorticoids, their esters are synthesized. E.g. the hydrocortisone water-insoluble ester, particularly hydrocortisone cypionate, is used orally in doses expressed in terms of hydrocortisone for slower absorption from the gastrointestinal (GI) tract. The extremely water-soluble 21-sodium succinate and 21-sodium phosphate esters are used for i/m injection in the management of emergency conditions that can be treated with anti-inflammatory steroids. The phosphate ester is completely and rapidly metabolized by phosphatases, with a half-life of less than 5 minutes. Phosphate ester display similar rapid action in case of inhalation or intranasal application. The sodium succinate ester is slowly and incompletely hydrolyzed, and peak hydrocortisone levels are attained in 25-35 minutes. Hydrocortisone butyrate, hydrocorti-sone buteprate, and hydrocortisone valerate are used topically.

Fig. 11. Hydrocortisone esters

Substituents added to the cortisol molecule may alter bioactivity and receptor binding affinity independently of other functional groups present on the molecule. While a high receptor binding ability does not necessarily reflect overall pharmacological activity, it is an important factor in glucocorticoid drug design. Two of these important modifications are as follows:

1. Halogenation. Halogenation is preferable in 6,7,9,12 positions. Insertion of fluorine in 9α position leads to fludrocortisone creation, which is only about 10 times more active than its parent compound (cortisol), but its mineralocorticoid activity is 300–600 times greater. This is undesirable 177 since it leads to edema; thus, the compound application is limited. It is important to note that 9- bromine derivative has half of cortisol activity; it means that glucocorticoid activity is inversely proportional to the atom radius present in the 9th position.

2. Additional double bonds. Δ1-compounds (where Δ1 indicates the position of a double bond (1- 2) were introduced, like prednisone, a Δ1-1l-ketone, and prednisolone, its 11-hydroxy analogue. Changing the geometry of the A ring (fig. 10) increased the potency (they are 3-4 times more active than cortisol) without augmenting mineralocorticoid activity. Besides, these derivatives have longer half-life time than hydrocortisone, as the ring A is metabolized difficultly. If the double bond is inserted in 9-11 position and the 11 position doesn’t contain an oxygen substituent, then glucocorticoid activity increases. Cortisol is an exception is; in this case activity decreases.

The introduction of a methyl group in methylprednisolone resulted in a slight increase in activity (without augmenting mineralocorticoid activity); however, the greatest improvements in activity came from the combination of a double bond, halogen, and methyl substituents.

Further modifications, in which two approaches were combined, lead to creation of compounds having 9α-fluoro group in addition to Δ1 unsaturation. Because of fact that these modifications enhance not only glucocorticoid, but also mineralocorticoid activity, an additional 16α-OH was inserted. Triamcinolone was created. This preparation is similar to prednisolone by its anti- inflammatory action, but lacks the mineralocorticoid activity.

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At the same time this preparation oral bioavailability is lower because lipophilicity is decreased due to hydrophilic group insertion, on the other hand, reduced its metabolic stability.

Injection farmaceutical forms for this preparation were created (IM) as well as prolonged action esters diacetate (1 to 8 weeks) and its hexacetonide (3 to 4 weeks). Triamcinolone doesn’t have mineralocorticoid effect, it also may induce sodium and water diuresis, and causes other unwanted side effects.

16th hydroxyl substitution by methyl group led to the development of dexamethasone, which was one of the most active and stabile glucocorticoids.

This structural change has goal to stabilize 17α-keto group toward the metabolic changes and increase bioavailability. Unlike16-hydroxylated derivatives, a methyl group increases the anti- inflammatory activity by increasing lipophilicity and, consequently, receptor affinity; but this group also decreases minerealocorticoid activity. In result of this, the activity of dexamethasone is 20 times greater than that of hydrocortisone and 5 times the anti-inflammatory activity of prednisolone.

Dexametazone

Betamethasone differs from dexamethasone only in configuration of the 16-methyl group-in this case β. It has similar activity to dexametasone, although in some cases even greater activity and it has been reported to be less toxic than other steroids, however, side effects typical to other glucocorticoids may occur with prolonged use. Beclomethasone, a 9α-chloro analogue of betamethasone, is topically-used for dermal, inhalation or intranasal therapy. For its’ systematic glucocorticoid activity it inferiors to fluorine derivatives, but locally, in dipropionate form it 500 times greater than betamethasone or dexamethasone, and approximately five times greater than

179 fluocinolone acetonide or triamcinolone acetonide. Another potent topical glucocorticoid (2 fluor derivative).

Glucocorticoid preparation treatment pathway depends on the type of disease, as well as drug's pharmacological, pharmacokinetic and physico-chemical properties. According to actiion glucocorticoids are systemic (oral, parenteral), dermal, inhalation and intranazal.

For the dermal or inhalation, pharmaceutical forms there can be used systemic as glucocorticoids derivatives: triamcinolone acetonide, betametazone dipropionate, as well as special synthesized glucocorticoids fluocinolone acetonide, aclometasone, beclometazone, fluticasone propionate etc.

There is a halogen atom in the majority of this glucocorticoids: fluor (di- or mono-fluorinated derivatives) or there is special cyclic ketal group in their structure.

There are also derivatives, which doesn’t contain halogen atom, but have expressed activity, which is conditioned by the mentioned group, e.g. desonide, or amcinonide, which, although contains one halogen atom, its activity is high due to more lipophilic cyclopentanone ketal and 21- acetate group.

Inhalation used glucocorticoids aren’t lack of systemic effect, consequently side effects, as far as in the nose cavity and lungs blood supply is good and they can be easily absorbed, consequently these preparations design should provide the following pharmacokinetic properties: they should rapidly undergo first pass metabolism at the systematic use, should have short half-life time and easily 180 metabolized to inactive metabolites, and at the same time have affinity to the glucocorticoid receptors. This will provide that they quickly reach to the receptor in the applied place and have influence, in case of absorption in the body quickly undergo inactivation not leaving systemic effect. Structural some changes led to new steroids creation, which in contrast to older preparations have greater affinity to the receptor and large therapeutic index, at the same time low bioavailability: mometazone furate, budesonide and fluticasone propionate.

This compounds are very lipophilic due to which better penetrate to the target using locally and bind with the receptor with great affinity. However, their bioavailability is 1% (mometasone) at the systematic intake, because during first pass metabolism they are easily metabolized to inactive metabolites.

FEMALE SEX HORMONES

The naturally occurring progestins are C21steroids and contain a 3-keto-4-ene structure in the A ring and a ketone at the C21position. The most potent endogenous progestin is progesterone. The class of steroids that contains the male sex hormones is the androgen class. In female organism are present androgens too, but the production and circulating plasma levels of estrogens and progestins are substantially higher in females.

Female sex hormones are specific steroids necessary for reproduction as well as for the development of secondary sex characteristics. They are comprised of two classes—estrogens, progestins. The naturally occurring estrogens are C18 steroids and contain an aromatic A ring with a hydroxyl group at the 3 position. The most potent endogenous estrogen is estradiol.

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O OH

HO O

Estradiol Progesterone

Steroid hormones are synthesized from cholesterin in the body.

Sex hormones biosynthesis

Estrogenes

Three endogenous estrogens are present in women.

 Estradiol, the most potent of the three, represents 10 to 20% of the circulating estrogen.  Estrone is 10-fold lesspotent than estradiol and accounts for 60 to 80% of the circulating estrogen. 182

 The remaining 10 to 20% is in the form of estriol, a very weak estrogen.

Estradiol plays a key role in several physiological processes, including the development of secondary sex characteristics during puberty, stimulation of the mammary glands during pregnancy, and thermoregulatory capacity.

Metabolism

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Metabolism of the estrogens to their water-soluble glucuronide conjugates occurs mainly in the liver. They are oxidized at16th position and converted to estriol which is eliminated by urine in glucuronide form. Estrogens are metabolized by estrogen 2/4-hydroxylase(CYP3A4) at positions ortho (2th and 4th) to the 3-phenolic group to form the 2-hydroxyestrogens and the 4- hydroxyestrogens. The resulting catechol estrogens bind to estrogen receptors (ERs) and produce weak to moderate estrogenic effects. These metabolites are unstable in vivo, however, and are rapidly converted to their 2-methoxyand 4-methoxyestrogen metabolites as well as to their glucuronide, sulfate, and glutathione conjugates.

Mechanism of Estrogenic Action

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Estradiol facilitates these processes via its biological target, the ER, of which there are two subtypes (ER-α and ER-β). The two receptors differ in size: ER- α is composed of 595 amino acids, and ER- β is composed of 485 amino acids. The expression and distribution of these subtypes is inconsistent between the various tissues and organs, which may explain the wide response that is observed. The predominant ER in the female reproductive tract and mammary glands is ER- α, whereas ER- β is found primarily in vascular endothelial cells, bone, and male prostate tissues. Expression of both ER-α and ER-β can be regulated hormonally via estradiol. Estradiol has similar affinities for both ER-α and ER-β can, which is not the case for certain non-steroidal estrogenic compounds and antiestrogens.

Within each target cell are two receptor locations, one within the nucleus, where a genomic mechanism pre-dominates, and a second within the cell membrane, where a non genomic mechanism prevails. Unlike the nuclear receptors, the cell membrane receptors are coupled to G proteins that are linked to a cascade of intracellular signals. When estradiol binds either ER-α or ER- β, the receptor protein is phosphorylated and undergoes a conformational change to produce dimmers. The dimeric ER complex then migrates from the cytosol to the cell nucleus, where it teams up with specific estrogen-response elements (ERE) found within an adaptor protein, typically a promoter, which aids in binding of the complex to estrogen activated genes. Before binding to ERE it forms transcription unit. If transcription unit contains coactivator (CoA), it regulates DNA transcription. When transcription unit contains CoR repressor molecule it forms antiestrogen complex and prevents DNA transcription.

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The ER is divided into six regions A–F. The DNA-binding domain (region C) is essential for the interaction of the ER with an estrogen-response element. The ligand-binding domain (region E) is the site of estradiol binding and the site of competitive binding by antiestrogens. The activating function (AF-1 and AF-2) regions are the areas of the ER that interact with coactivator molecules to form an effective transcription unit at an estrogen-responsive gene. The ligand binding domain (region E) is the site of estradiol binding and the site of competitive binding by antiestrogens. For example, AA 351 position is the site for antiestrogen drug raloxifen binding. The activating function (AF-1 and AF-2) regions are the areas of the ER that interact with coactivator and corepressor molecules to form an effective transcription unit at an estrogen responsive gene.

Estradiol is one of the endogen ligand for estrogen receptors. The intramolecular interactions between receptors and endogen ligand are the following:

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 3-hydroxyl group of estradiol forms hydrogen bond with receptor arginine residue, and ion- dipol interaction with receptor glutamine acid residue as well,  - interaction is formed between ligand aromatic ring and receptor phenylalanine aromatic ring  Estradiol forms extra hydrogen bond with receptor histidine residue due to 17-hydroxyl group which provides strong interaction with receptor. Thus, pharmacophor groups for estrogen compounds are phenolic hydroxyl and 17β-groups and the distance between these groups should be aproximetally 11 (10,3-12,1) A for binding to receptors.

Steroidal Estrogens

Estradiol

Estradiol is the most potent endogenous estrogen, exhibiting high affinity for the ER and high potency when administered parenterally. When administered orally, estradiol is promptly conjugated in the intestine and oxidatively metabolized by the liver, resulting in its low oral bioavailability and therapeutic effectiveness.

Ethynyl estradiol and mestranol.

One method to increase the oral bioavailability of estradiol is to prevent metabolic oxidation of the estradiol C-17hydroxyl group to estrone. This is readily accomplished via alkylation of the C- 17position with a chemically inert alkyne group (e.g., ethynyl estradiol). This synthetic analogue is several hundred-fold more potent than estradiol. Another semisynthetic estrogen, mestranol, is the 3- O-methyl ether of ethynyl estradiol. Mestranol is a prodrug and, following oral administration, is rapidly metabolized to ethynyl estradiol via hepatic O-demethylation.

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Esters of estradiol.

To deliver estrogens with a longer duration of action, 17β-estradiol must be derivatized into an ester prodrug. In contrast to the ethynyl derivatives delivered orally, these estrogen analogues usually are administered intramuscularly. Slow hydrolysis of the ester releases free estradiol over a prolonged period of time. The therapeutically useful esters of estradiol include 17β-valerate and 17β- cyclopentylpropionate (cypionate). Estradiol cypionate is available as a sterile solution of the drug in oil (e.g., cottonseed oil), with a duration of action of 14 to 28 days. Estradiol valerate is available as a sterile solution in a vegetable oil (e.g., sesame oil or castor oil), with a duration of action of 14 to 21 days.

Other types of highly active, orally bioavailable estrogen analogues include those with a labile ether (e.g., 3-(2-tetrahydropyranyl) and 17β-(2-tetrahydropyranyl) estradiol). These drugs proved to be 12- and 15-fold as active, respectively, as estradiol.

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Nonsteroidal Estrogens

Stilbene derivatives

The steroid nucleus is not required for estrogenic action. Several derivatives of stilbene (diphenylethylene) that were used therapeutically demonstrate potent estrogenic activity. Diethylstilbestrol (DES), prepared in 1939, was one of these stilbenes. Diethylstilbestrol has 10-fold the estrogenic potency of its cis isomer, largely because the trans isomer more closely resembles estradiol. Unfortunately, when DES was administered to relieve pregnancy related symptoms, it was correlated with abnormal growth in the offspring.

Structure–Activity Relationships

As a result of numerous studies, there is an extensive body of knowledge regarding structure– activity relation-ships for estrogens.

 The aromatic A ring and the C-3-hydroxyl group are structural features essential for estrogenic activity.  The 17-hydroxyl, the distance between the C-3 and C-17-hydroxyl groups, and the presence of planar hydrophobic scaffolding also are important structural contributors and help to optimize estrogenic activity. Ideally, the distance between the oxy-gen atoms of the C3and C17hydroxyl groups should range from 10.3 to 12.1 Å.  Substitution of the estrogen steroid nucleus can significantly modify estrogenic activity. Functionality at the C-1-position greatly reduces activity, and only small group scan be accommodated at the 2 and 4 positions.  Addition of hydroxyl groups at positions 6, 7, and 11 reduces activity.  Removal of the oxygen function from position 3 or17, or epimerization of the 17-hydroxyl group of estradiol results in an estrogenic analogue that is less active.  The equine estrogens contain one or two additional double bonds in the steroidal B ring (equilin and equilenin, respectively). The presence of this unsaturation substanially boosts the estrogenic potency of these estrogens.

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 Certain modifications at the 17 and 16 positions can lead to enhanced activity. For example, the 17-ethynyl or 17-vinyl groups provide the greatest activity, where as highly polar substituents at this position are poorly tolerated.

 Orally bioavailable estrogen analogues 3-(2-tetrahydropyranyl) and 17β-(2-tetrahydropyranyl) estradiol are highly active and esters have long action duration.

Antiestrogen drugs and SERM preparations

Antiestrogen drugs and SERM (selective estrogen receptor modulator) preparations are used as contraceptivs and as anticancer drugs in hormon releted cancers. SAR in triphenylethylen derivatives group

Antiestrogen compounds are formed based on nonsteroidal estrogen structures related to the stilbene family. Etamoxytriphetol was created as a preparation.

Unfortunately, clinical researches have shown that low concentration has low efficiency, but high dosage brings to numerous side effects. In the search for more potent antiestrogens, clomiphene was identified as an effective post coital contraceptive. It was created by insertion of double bond and chlorine atom in etamoxytriphetol molecule. Clomipfen is a mixture of cis (E) and trans (Z) isomers and is used for ovulation induction.

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Moreover, trans (Z) isomer (Zuclomiphene) has estrogenic activity and cis (E) isomer (Enclomiphen) shows partial agonist activity.

In order to decrease side effects chlorine atom was replaced by alkyl chain and the formed preparation was Tamoxifen wich is used in brest cancer. It also is like clomifen, a racemic mixture of cis and trans isomers, one of which has estrogen activity, and the second shows antiestrogen activity. This drug is administered as the Z-diastereomer and, in some countries, is used for ovulation induction. Rigid analogue of tamoxifen, nafoxidine was syntesised which has antiestrogen activity but was toxic for human body. Keto group was inserted in the center of Nafoxidine structure in order to decrease toxicity. The ptreparation is Trioxifen, but in clinic it doesn't used as an anticancer drug.

Based on trioxifen structure raloxifen was created, which became the SERM (selective estrogen receptors modulators) of the first generation. This preparations show tissue specific estrogen agonist/antagonist properties. Raloxifen has high affinity towards estrogen receptors, as well as some

191 anticancer activity in laboratory animals. In clinic raloxifen is used for prevention of osteoporosis and breast cancer.

Raloxifen, which is the rigid analoge of tamoxifen, belongs to benzothyophen derivatives group and like tamoxifen is a partial agonist. Du to this it has agonist activity on bones and cardiovascular system and antagonist activity on breast tissue receptors.

ARZOXIFENE. Arzoxifene is a third-generation SERM currently in Phase III clinical trials for the treatment of ER-positive recurrent/metastatic breast cancer. It was created based on raloxifen structure (the keto group in raloxifen was replaced by ether group). Similar to raloxifene, it is an ER antagonist in both breast and uterine tissues and an ER agonist on bone and the cardiovascular system. Arzoxifene is able to both preserve and build bone mineral density, which makes it a viable candidate for the treatment of osteoporosis. Clinical trials indicate that arzoxifene also can be utilized as a chemopreventive agent against breast cancer.

Arzoxifene

Aromatase inhibitors

Androstenedione Derivatives. Inhibitors of aromatase block the conversion of androgens to estrogens and, therefore, have the therapeutic potential to control reproductive functions and aid in the treatment of estrogen-dependent cancers, such as breast cancer.

These steroidal agents compete with androstenedione for the active site of the aromatase enzyme. The drug of this group is Exemestane.

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Triazole Derivatives. Triazole based aromatase inhibitors were developed based on the aromatase inhibitor aminoglutethimide and include anastrozole and letrozole (3. The triazoles inhibit aromatase as a result of the N-4 nitrogen of the triazole ring interaction with the heme iron atom of this CYP19enzyme complex. Anastrozole and letrozole are competitive inhibitors of aromatase and selectively inhibit the conversion of testosterone to estrogens in all tissues.

Anastrozole Letrozole

Progestins

The naturally occurring progestins are C21steroids and contain a 3-keto-4-ene structure in the A ring and a ketone at the C21position. The most potent endogenous progestin is progesterone.

Metabolism

Progesterone is rapidly metabolized by the liver. Progesterone is mainly excreted renally as the glucuronide and sulfate conjugates of 5β-pregnanediol. The formation of 5β-pregnanediol from progesterone is characterized by reduction of the 4,5-double bond and reduction of the 3-ketone. Other routes of metabolism include 6α-hydroxylation and reduction of the 20-ketone. These metabolites have no significant progestogenic activity.

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Metabolism of progesterone

Progesterone and its derivatives

Progesterone has a significant role in priming the uterine endometrium for implantation of a potential blastocyst. It also is involved in formation of the placenta post implantation, the development of mammary glands, and by preventing contraction of the uterine musculature, pregnancy maintenance. Progesterone also has inhibitory roles, including ovulation prevention via an antigonadotropic effect and inhibition of the conversion of testosterone to dihydrotestosterone, an active metabolite, by virtue of its ability to be a substrate for 5α-reductase. Interestingly, progesterone reduces nuclear estradiol receptor levels and induces 17-hydroxysteroiddehydrogenase, the enzyme that catalyzes the conversion of estradiol to the less potent estrone. There are some limitations as to how progesterone can be administered, because it has relatively low bioavailability when administered orally. To achieve consistent therapeutic benefit, progesterone must be administered either by injection or intravaginally.

Dihydrogesterone is similar to progesterone by its structure and pharmacological action. It contains double bond at 6,7 position, has high affinity to progesterone receptors and absorbed rapidly.

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As a result, development of orally active derivatives has been a significant priority.

Synthetic Progestins

Early generations of the progestins were utilized primarily for contraceptive purposes, so antigonadotropic activity was considered to be desirable. Unfortunately, many of these agents were plagued by androgenic activity and the corresponding adverse effects. Development of newer progestins is now focused on analogues with improved progesterone receptor selectivity and little or no effect on the androgen, estrogen, or glucocorticoid receptors. From a structural perspective, these synthetic progestins contain either a pregnane or androstane steroid nucleus

The synthetic progestins generally can be divided into three classes of compounds: analoges of 17α-hydroxyprogesterone, androstane, 19-norprogesterone.

17α-hydroxyprogesterone androstane

19-norprogesterone

Derivatives of 17α-hydroxyprogesterone

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Medroxyprogesterone acetate

The initial structural modifications made to progesterone led to only weak active or inactive analogues. For example, 17α-acetoxy progesterone had limited activity when administered orally. Further structural modification of 17α-acetoxy progesterone was aimed at limiting metabolic hydroxylation at C6. This was accomplished by the addition of a C6 substituent, and the resulting analogue displayed improved biological activity. Among the first of these substituted 17α-acetoxy progesterone analogues to be utilized therapeutically was medroxyprogesterone acetate, a 6α-methyl progesterone analogue. This analogue is 25-fold more active than ethisterone. Following oral administration, medroxyprogesterone acetate is completely and rapidly deacetylated by first pass metabolism to medroxyprogesterone. Medroxyprogesterone is extensively metabolized via pathways similar to those for progesterone, except for 6α-hydroxylation. Most medroxyprogesterone acetate metabolites are excreted in the urine, primarily as glucuronide conjugates.

Megestrol acetate

Progestin activity is further enhanced when a double bond is introduced between positions 6 and 7, as is found in megestrol acetate. Megestrol is used primarily in the treatment of breast and endometrial carcinomas and in postmenopausal women with advanced hormone-dependent carcinoma. Less than 10% of an oral dose undergoes metabolism. Several major metabolites appear in the urine (e.g., 2-hydroxy and 6-hydroxymethylmegestrol and their glucuronide conjugates).

Derivatives of androstane

17α-ethynil, ethyl, methyl substituents of androstane derivatives possess high oral bioavailability. Closely related to testosterone, this progestins has significant androgenic activity.

Ethisterone and its analogues

Ethisterone a 17α-ethynyl derivative of testosterone, is one of the first synthetic progestins to be used therapeutically. In 1937, this agent was synthesized from male sex hormones (androstanes) in an attempt to find an orally active androgen. Ethisterone later proved to be an effective oral progestin and became useful in the treatment of menstrual dysfunctions. Several molecular modifications of ethisterone have improved progestogenic action, including introduction of methyl groups in the C6α

196 and C21positions (e.g., dimethisterone). A second breakthrough was made in 1944, when Ehrenstein (54) discovered that the C19 methyl group is not necessary for progestogenic activity. In fact, this work showed that loss of the C-19-methyl results in analogues with activity equal to or greater than that of parenterally administered progesterone. In 1953, Djerassi et al. synthesized 19- norprogesterone. This drug differed from the natural hormone only in replacement of the C19 angular methyl group with a hydrogen atom. When administered parenterally, this analogue was eight-fold more active than progesterone and, at the time, was the most potent progestin known. It’s the first androstane which shows 1/3 activity of progesterone in case of subcutaneous administration and 15-fold more potent in per oral administration.

Derivatives of 19-norprogesterone

A second breakthrough was made in 1944, when Ehrenstein discovered that the C19-methyl group is not necessary for progestogenic activity. In fact, this work showed that loss of the C19- methyl results in analogues with activity equal to or greater than that of parenterally administered progesterone. In 1953, Djerassi et al. synthesized 19-norprogesterone. This drug differed from the natural hormone only in replacement of the C19-angular methyl group with a hydrogen atom. When administered parenterally, this analogue was eight-fold more active than progesterone and, at the time, was the most potent progestin known.

19-norprogesterone

Norethindrone (norethisterone) and norethynodrel Research on 19-norsteroids as potential progestins culminated in the synthesis of two potent, orally active progestins, norethindrone and norethynodrel. These two substances were among the first 19-norsteroids to be used clinically for progesterone-related disorders. The activity of norethindrone is increased further by the addition of a chlorine substituent at position 21 (blocks metabolic hydroxylation) or by the addition of a methyl group at carbon 18 (norgestrel). Acetylation of the 17β-OH of norethindrone increases the duration of action of the drug (norethindrone acetate). Removal of the 3-keto function of norethindrone allows retention of potent progestin activity and no androgenic effects. Change of 3-keto group by acetate group leads to more orally active progestin than progesterone (ethinodiol diacetate).

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Following oral administration, norethindrone acetate is completely and rapidly deacetylated by hepatic and intestinal first-pass metabolism to norethindrone, with an oral bioavailability of approximately 64%. Subsequent metabolism of norethindrone includes reduction of the Δ4 double bond and keto group.

norethindrone norethindrone acetate

norethynodrel ethinodiol diacetate

Norgestrel and levonorgestrel

Norgestrel is formulated as a racemic mixture despite the fact that only its levo isomer, levonorgestrel, is pharmacologically active. Levonorgestrel exhibits some androgenic activity but no glucocorticoid or antimineralocorticoid action. The oral bioavailability of levonorgestrel is approximately 95%. Levonorgestrel undergoes metabolic reduction of its ketone and is hydroxylated.

Levonorgestrel

Norgestimate and desogestrel

Norgestimate is considered to be a pro-progestin (prodrug), because it rapidly undergoes a two- step metabolic transformation to form two active products, norelgestromine (levonorgestrel 3-oxime) and levonorgestrel. Deacetylation occurs in the intestine and liver, whereas conversion of the 3- oxime to the corresponding ketone occurs primarily in the liver. Norgestimate exhibits high selectivity for the progesterone receptor and low androgenic activity.

Desogestrel also is a prodrug and is rapidly metabolizedin the intestinal mucosa and on first pass through the liverto its active metabolite, etonogestrel (3-ketodesogestrel). Following oral administration, the relative bioavailability for desogestrel is approximately 84%. Desogestrel also exhibits high selectivity for the progesterone receptor and low androgenic activity.

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Norgestimate Desogestrel

Gestodene

Unlike the other members of the third generation of progestins, gestodene is not a prodrug. It exhibits nearly 100% oral bioavailability and excellent receptor binding affinity for the progesterone receptor.

Gestogene

Dienogest

Classified as a testosterone-like progestin, dienogest is structurally unique in that it contains an estrane skeleton, a C17-cyanomethyl group, and 9unsaturation.

Dienogest

Elcometrine is a member of the 19-norprogesterone class of progestins. The C16-methylene functionality substantially increases its affinity for the progesterone receptor. Elcometrine does not bind to the androgenic receptor and, therefore, does not possess either androgenic or antiandrogenic activity. Unfortunately, like medroxyprogesterone acetate, elcometrine has affinity for the glucocorticoid receptor.

Eclomdetrine

Nomogestrol acetate is structurally similar to megestrol acetate but lacks the angular C19-methyl group. It has substantially better selectivity for the progesterone receptor and higher potency than medroxyprogesterone acetate. Nomogestrol acetate has potent antigonadotropic action. It exhibits no glucocorticoid, antimineralocorticoid, or androgenic activity.

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Nomogestrol acetate

Trimegestone, the most potent of the 19-norproges-terones, contains an unusual C21-hydroxyl group. It has very high affinity for the progesterone receptor but only weak affinity for the mineralocorticoid receptor. Trimegestone displays no glucocorticoid, androgenic, or antiandrogenic action. This progestin undergoes metabolic hydroxylation to produce metabolites with substantial progestogenic action.

Trimegestone

Derived from spironolactone, drospirenone is the only progestin with antimineralocorticoid activity. Its affinity for the mineralocorticoid receptor is fivefold greater than that of aldosterone. Drospirenone also has antiandrogenic action, because it blocks testosterone from binding to androgenic receptors but does not exhibit estrogenic or glucocorticoid receptor activity. This androstane-based progestin has several distinctive functional groups: two cyclopropyl groups, one that includes C6 and C7 and the other C15 and C16, and a C17 lactone.

Drospirenone

Progesteron ereceptor modulators

Like the SERMs, the selective progesterone receptor modulators bind to the progesterone receptors and act as either an agonist or antagonist depending on the absence or presence ofprogesterone and the tissue that is being targeted. In the absence of progesterone, these agents demonstrateprogestin activity. In the presence of progesterone, they exhibit antiprogestin activity in some target tissues, particularly the endometrium. Several investigational agents, including asoprisnil, have suppressed the growth of endometrial implants while estrogen concentrations remained at similar levels to those found during the follicular phase of the menstrual cycle. Asoprisnil undergoes metabolic O-dealkylation to an active metabolite.

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Asoprisnil

Progestin Antagonists

Mifepristone. An antiprogestin is a substance that competes with progesterone for its receptor and, ultimately, prevents progesterone from binding to and activating its receptor. Mifepristone is metabolized primarily via CYP3A4 pathways involving mono- and di-N-demethylation and terminal hydroxylation of the 17-propynyl chain. Mifepristone also demonstrates antiglucocorticoid activity.

Additional antiprogestin analogues, such as onapristone, exhibit less antiglucocorticoid activity. These antiprogestins also have demonstrated therapeutic potential for the treatment of hormone- dependent breast cancer.

Mifepristone Onapristone

Metabolic demethylation of onapristone

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HORMONES OF THE THYROID GLAND

Thyroid hormones are iodinated amino acids dеrived from L-tyrosine. Two active hormones are synthesized in the thyroid gland- T4 (thyroxin) and T3 (trioidthironine). The thyroid gland also contains two quantitatively important iodinated acids, diiodo L-thirosine (DIT) and monoiodo L- thirosine (MIT).

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Biosynthesis of Thyroid Hormons

The synthesis of the thyroid hormones, T3 and T4, is regulated by thyrotropine (thyroid stimulating hormone TSH), which stimulates the synthesis of thyroglobulin, thiroperoxidase (TPO) and hydrogen peroxyde. The formation of the thyroid hormones depends on an exogenous supply of iodide. The formation of the thyroid hormones involves the following complex sequence of events:

1. active uptake of iodide by the follicular cells,

2. oxidation of iodide and formatioa of iodothyrosyl residues of thyroglobulin,

3. formation of iodothyronines from iodothyrosines,

4. proteolysis of thyroglobulin and release of T4 and T3 into blood,

5. conversion of T4 to T3.

The first step in the synthesis of the thyroid hormones is the uptake of iodide from the blood by the thyroid gland. The mechanism enabling the thyroid gland to concentrate blood iodide against a gradient into the follicular cell is the iodide pump (NIS, sodium/iodide symporter). In the apical 203 membrane iodide is oxydized by TPO in the presense of hydrogen peroxide to an active iodine species that, in turn, iodinates selected thyrosileresidues of thyroglobulin.

2J+ H2O2+ 2H+= 2J++ 2H2O

Consistent with the conditions necessary for the aromatic halogenation, the iodination of the thyrosil residues. The iodinating species is thought to be hypoiodate (OI-). After that coupling of iodothyrosil residues takes place at thyroglobulin and involves the coupling of the two outer rings from DIT residues to become T4, whereas the coupling of the oputer ring from MJT with DIT results in the formation of T3.

In response to demand for thyroid hormones, the release of thyroid hormones from thyroglobulin begins with the its resorption of thyroglobulin via endocytosis into the follicular epithelial cells and its subsequent complete proteolysis by the lysosomal digestive enzymes of the follicular cells.

Thyroglobulin proteolysis yields MIT, DIT, T3, and T4. Although MIT and DIT are formed, they do not leave the thyroid but, instead, are selectively deiodinated to tyrosine and recycled into new thyroglobulin. The iodide is recycled into hypoiodite for subsequent iodination, conserving the essential nutrients for the thyroid gland. Both T3and T4are secreted by the cell into the circulation. A defect in the cyclization of MIT and DIT can lead to hypothyroidism and goiter by increasing their elimination in the urine.

T4 usually is considered to be a prohormone. The enzymatic conversion of T4 to T3 is an obligate step in the physiological action of thyroid hormones in most extrathyroidal tissues. In the peripheral tissues, approximately 33% of the T4 secreted undergoes deiodination to give T3' and another 40% undergoes deiodination of the inner ring to yield the inactive material rT3.The deiodination of T4 is a reductive process catalyzed by a group of enzymes named iodothyronine deiodinases, referred to as deiodinases and symbolized by D, found in a variety of cells. Approximately 80% of the T3 is derived from circulating T4'. Three types of deiodinases are currently known, and these are distinguished from each other primarily based on their location, substrate preference, and susceptibility to inhibitors. Type I deiodinase is found in liver and kidney and catalyzes both inner ring and outer ring deiodination (i.e., T4 to T, and rT3 to 3,3'-T2). Type II of deiodinase catalyzes mainly outer ring deiodination (i.e., T4 to T3 and T3 to 3,3'-T2) and is found in brain and the pituitary. Type III deiodinase is the principal source of rT3 and is present in brain, skin, and placenta.

The iodothyronines secreted by the thyroid gland into thyroid vein blood are of limited solubility. They equilibrate rapidly, however, through noncova1ent association with three major binding proteins: thyroid binding globulin (TBG), transthyretin (TIR; formerly called T4 binding prealbumin), and albumin. Thyroid binding globulin is the primary serum binding protein because of its higher affinity for T4. Under normal conditions, 75% of T4 is bound to TBG, 10 to 15% to TIR, and

5 to 15% to albumin. When bound, T4 is not physiologically active but does provide a storage pool of

204 thyroid hormone, which can last 2 to 3 months (mean half-life of T4, 6.7 days in adults). The plasma proteins involved in thyroid hormone transport and their approximate association constants (K.) for

To and T4 are shown in Table 34.l. This table indicates that TBG has a high affinity for T4 (K; - 1010

M) and lower affinity for T3. The lower binding affinity for T3 toplasma proteins may be an important factor in the more rapid onset of action and the shorter biological half-life for T3.

Metabolism and Excretion

As discussed earlier, T4 is considered to be a prohormone, and its peripheral metabolism occurs in two ways: outer ring deiodination by the enzyme 5'-D, which yields T3, and inner ring deiodination by the enzyme 5-D, which yields rT3' for which there is no known biological function (Fig. 34.4). In humans, deiodination is the most important metabolic pathway of the hormone, not only because of T4 activation and inactivation process, but also in quantitative terms. Degradative metabolism of the thyroid hormones, apart from peripheral deiodination, occurs mainly in the liver, where both T3 and

T4 are conjugated to form either glucuronide (mainly T4) or sulfate (mainly T3) with the phenolic hydroxyl group. The resulting iodothyronine conjugates are excreted via the bile into the intestine, where a portion is hydrolyzed by bacteria. The conjugates also undergo marginal enterohepatic circulation and are excreted unconjugated in feces. T4 is conjugated with sulfate in kidney and liver, and T4-4'-O-sulfate, an excellent substrate for 5'-D (15), is believed to play a role in the regulation of T, metabolism. Additional metabolism, involving side-chain degradation, proceeds by transamination, oxidative deamination, and decarboxylation to yield thyroacetic acid and thyroethanediol; cleavage of the diphenyl ether linkage has been detected as well, both in vitro and in vivo. The reactions through which thyroid hormone is metabolized are summarized in Figure 34.5.

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Biochemical functions

The thyroid hormones, T4 and T3, play numerous, profound roles in regulating metabolism, growth, and development and in maintaining homeostasis. Their reactions and products influence carbohydrate metabolism, protein synthesis and breakdown, and cardiovascular, renal and brain function. It generally is believed that these actions result from effects of thyroid hormones on protein synthesis.

Thiroid hormones preparations

Thiroid hormones preparations are natural and syntetic. Natural remedies are obtained from animal thyroid gland. As a syntetic preparations are used T4 (levothyroxine), T3 (liothyronine), dT4

(dextrothyroxine), and T4-T3 mixtures (Liotrix), Thyreocomb (mixture of T4, T3 and potassium iodide). There are used in hypothyroidism.

Structure-Activity Relationships of Thyroid Analogues

The structure–activity relationships of thyroid hormones and related structural analogs have been studied using both qualitative and quantitative methods, including the Hansch correlation. The synthesis and biological evaluation of a wide variety of T4 and T3 analogues allowed a significant correlation of structural features with their relative importance in the production of hormonal responses. The SARs are discussed in terms of single structural variation of T4 in the 1) alanine side chain, 2) 3- and 5-positions of the inner ring, 3) the bridging atom, 4) 3'- and 5'-positions of the outer ring, and 5) the 4'-phenolic hydroxyl group.

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Aliphatic Side Chain

The naturally occurring hormones are biosynthesized from L thyrosine and possess the L-alanine side chain.

1. The L isomers of T4, and T3 (compounds 1 and 3 in Table 34.2) are more active than the D- isomers (compounds 2 and 4).

2. The carboxylate ion and the number of atoms connecting it to the ring are more important for activity than is the intact zwitterionic alanine side chain.

3. In the carboxylate series, the activity is maximum with the two-carbon acetic acid side chain (compounds 7 and 8) but decreases with either the shorter formic acid (compounds 5 and 6) or the longer propionic and butyric acid analogues (compounds 9-12).

4. The ethylamine side chain analogues of T4 and T3(compounds 13 and 14) are less active than the carboxylic acid analogues.

5. In addition, isomers of T3 in which the alanine side chain is transposed with the 3- iodine or occupies the 2-position were inactive in the rat outer test, indicating a critical location for the side chain in the I-position of the inner ring.

Alanine-Bearing Ring

The phenyl ring bearing the alanine side chain, called the inner or α-ring, is substituted with iodine in the 3- and 5-positoins in T4 and T3.

1. As shown in Table 34.2, removal of both iodine atoms from the inner ring to form 3',5'-T2

(compound 15) or 3'-T1 (compound 16) produces analogues devoid of T4-like activity, primarily because of the loss of the perpendicular orientation of diphenyl ether conformation.

2. Retention of activity observed at replacement of the 3 and 5- iodine atoms with bromine (compounds 17 and18) empties that iodine does not play a unique role in thyroid hormone activity. Moreover, a broad range of hormone activity found with halogen free analogues (compounds 19 and 20) indicates that a halogen atom is not essential for activity.

3. Substitution in the 3- and 5- positions by alkyl groups significantly larger and less symmetric than methyl groups, such as isopropyl and secondary butyl moieties, produces inactive analogues (compounds 21 and 22). These results show that 3,5-disubstitution by symmetric, lipophilic groups not exceeding the size of iodine is required for activity.

Bridging Atom

Several analogues have been synthesized in which the ether oxygen bridge has been removed or replaced by other atoms.

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1. The biphenyl analogue of T4, (compound 23 in Table 34.2), formed by removal of the oxygen bridge, is inactive in the rat antigoiter test. The linear biphenyl structure is a drastic change from the normal diphenyl ether conformation found in the naturally occurring hormones.

2. Replacement of the bridging oxygen atom by sulfur (compound 24) or by an ethylene group (compound 25) produces highly active analogues

Phenolic Ring

1. The phenolic ling, also called the outer or β-ring, of the thyronine nucleus is required for hormonal activity.

2. Variations in 3'- or 3',5'-substituents on the phenolic ling have dramatic effects on biological activity and affinity for the nuclear receptor.

3. The unsubstituted parent structure of this series L-T2(compound 26 in Table 34.2) possesses low activity.

4. Substitution at the 3'-position by polar hydroxyl or nitro groups (compounds 27 and 28) causes a decrease in activity as a consequence of both lowered lipophilicity and intramolecular hydrogen bonding with the 4'-hydroxyl.

5. Conversely, substitution by nonpolar halogen or alkyl groups results in an increase in activity in direct relation to the bulk and lipophilicity of the substituent-for example, F < CI < Br < I (compounds 29-31) and CH3 < CH2CH3 < CH(CH3h (compounds 32-34). Although 3'- isopropilthyronine (compound 34) is the most potent analogue known, being approximately 1.4 times as active as L-T3, n-propyltyironine (compound 35) is only about one-fourth as active as isopropyl, apparently because of its less compact structure.

6. As the series is further ascended, activity decreases with a further reduction for the more bulky 3'- phenyl substituent (compound 36).

7. Substitution in both 3'-and 5' -positions by the same halogen produces less active hormones (compounds 37 and 38) than the corresponding 3'-monosubstituted analogues (compounds 29 and 30). The decrease in activity has been explained as resulting from the increase in phenolic hydroxyl ionization and the resulting increase in binding to TBG (the primary carrier of thyroid hormones in human plasma).

Phenolic hydroxyl Group

1. A weakly ionized phenolic hydroxyl group at the 4`-position is essential for optimum hormonal activity.

2. Replecment of the 4`-hydroxyl with an amino group (compound 39) results in a substential decrease in activity, presumbly as a result of the weak hydrogen bonding ability of the latter group

3. The retention of activity observed with the 4`-unsubstituted compound (40) provides direct evidence for metabolic 4`-hydroxylation as an activating step. 208

4. Itroduction of a 4`-substituent that cannot mimic the functional role of a phenolic group, such as a methyl group (41), and that is not metabolically converted into a functional residue results in complate loss of hormonal activity. The thyromimetic activity of the 4`-methyl ether (42) was ascribed to the ready metabolic cleavage to form an active 4`-hydroxyl analogue. The pka of 4`- phenolic hydroxyl group is 6,7 for T4 (90% ionized at pH 7,4) and 8,5 for T3 (10% ionized). The greater acidity for T4 is reflective of iots stronger affinity for plasma protein and, consequently,of its longer plasma half-life.

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Key Points

The structural requirements for receptor binding, and therefore hormone activity, are:

1. Two aromatic rings perpendicular to each other and separated by a spacer atom (O, S, or C) that holds the rings at an angle of about 120°.

2. Halogen or methyl groups on the 3 and 5 positions of the ring that bears the alanine side chain (these substituents keep the rings perpendicular to each other and participate in hydrophobic bonding to the receptor).

3. An anionic side chain two or three carbons long, para to the bridging atom, forming an ion pair with the nuclear receptor (the -NH2 group decreases receptor affinity but plays a role in transport of the hormones and delays their metabolic degradation).

4. A phenolic 4'-OH group, which may be generated metabolically (e.g., by oxidation in vivo) if originally absent.

5. A lipophilic halogen, alkyl, or aryl substituent in the 3' position. An isopropyl group has the optimal effect.

6. A 5' substituent reduces activity in direct proportion to its size. It interferes with the binding of the 4'-OH group, increases the binding to transport proteins, and therefore reduces the concentration of available free hormone.

Antithyroid drugs for the treatment of hyperthyroidism

Iodide. Inhibition of the release of thiroid hormone by iodine is the basis for ist use in hyperthyroidism. Iodide decreases the vasculatory of the enlarged thyroid gland and it also might change the conformation of thyroglobulin, making the protein less susceptible to thyroidal proteolysis. Iodide, as Lugol’s solution (Strong Iodine Solution USP) or as saturated potassium iodide solution, is administered for approximately 2 weeks to ensure decreased vascularity and firming of the gland. Iodism, a side effect of iodine administration, is apparently an allergic reaction characterized by dermatological and common cold-like symptoms.

125J2 and 131J2 isotopes also are used, which destruct of the thyroid follicles.

Lithium salts have been used as safe adjuncts in the treatment of thyrotoxicosis Lithium is concentreted by the thyroid gland. Lithium ion inhibits adenylate cyclase, which forms cAMP. Formed in response to TSH, cAMP is a stimulator of the processes involved in thyroid hormone release from the gland. Inhibition of hormone secretion by lithium has proved to be a useful adjunct in treatment of hyperthyroidism.

Large anions such asthiocyanate (CNS-), isothiocyanate (SCN-), nitriles (RCN), TcO4

(pertechnetate) CIO4 (perchlorate) and thiooxazolidones. Thiocyanate is a large anion that competes with iodide for uptake by the thyroid gland; its goitrogenic effect can be reversed by iodide intake.

Goitrin, 5-R-vinyloxazolidine-2-thione, is a potent thyroid peroxidase inhibitor that is claimed to be more effective than PTU in humans and is held to be the cause of a mild goiter endemia in

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Finland. In rats, goitrin is actively taken up by the thyroid gland and appears to inhibit the coupling of thyroglobulin diiodotyrosyl residues.

Other compounds affecting thyroid function include sulfonamides, anticoagulants, and oxygenated and iodinated aromatic compounds. The hypoglycemic agent carbutamide and the diuretic Diamox are examples of sulfonamides. Of the anticoagulants, heparin appears to interfere with the binding of T4 to plasma transport proteins, but warfarin (Coumadin) and dicoumarol are competitive inhibitors of the substrate T4 or rT, in the 5'_ D reaction. Other oxygenated compounds affecting the 5'-D include resorcinol, long known to be a goitrogen, and phloretin, a dihydrochalocone with an IC5Qof 4 M.

The ability of oxidation products of 3,4-dihydroxycinnamic acid to prevent the binding of TSH to human thyroid membranes suggests that other oxygenated

phenols may interfere with thyroid hormone function in more than one way. Examples of iodinated drugs affecting thyroid function are the antiarrhythmic agent amiodarone and the radiocontrasting agents iopanoic acid and ipodoic acid. All of these compounds interfere with the peripheral deiodination of T4 and are being tested as adjuncts in the treatment of hyperthyroidism. Salicylates, diphenylhydantoin, and heparin are members of the large group competing with thyroid hormones for binding sites.

Methimazole, Propylthiouracil, and Related Compounds Thionamides are the most important class of antithyroid compounds in clinical practice used in ondestructive therapy of hyperthyroidism. These agents are potent inhibitors of TPO, which is responsible for the iodination of tyrosine residues of thyroglobulin and the coupling of iodotyrosine residues to form iodothyronines. These drugs have no effect on the iodide pump or on thyroid hormone release. The most clinically useful thionamides are thioureylenes, which are five- or six-membered heterocyclic derivatives of thiourea and include the thiouracil 6-n-propyl-2-thiouracil (PTU) and the thioimidazole 1- methyl-2-mercaptoimidazole methimazole, Tapazole, MMI). The uptake of these drugs into the thyroid gland is stimulated by TSH and inhibited by iodide.

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Thiouracil; R = H

Methylthiouracil; R = CH3

Propylthiouracil (PTU); R = n-C3H7 Methimazole(MMI. R = H)

Carbimazole (R = C2HsOCO)

Chemically, the grouping R-CS-N- as been referred to as thioamide, thionamide, thiocarbamide, or ifR is N, as it is in thiouracil, PTU, and MMI, it is called a thioureylene. This structure may exist in either the thioketo or thioenol tautomeric forms.

The study of 6-alkylthiouracil showed maximal anti-thyroid activity with 6-propylthiouracil. 6- Methylthiouracil has less than one-tenth the activity of PTU.

A number of studies have defined the structure-activity relationships (SAR) of the thiourac and other related compounds as inhibitors of outer ring deiodinase:

1. the C2 thioketo/thioenol group and an unsubstituted N1 position are essential for activity.

2. the enolic hydroxyl group at C4 in PTU and the presence of alky group at C5

and C6 enhance the inhibitory potency.

Methimazole has more TPO inhibitory activity and is longer-acting than PTU but, in contrast to PTU, is not able to inhibit the peripheral deiodination of T4, presumably because of the presence of the methyl group at NI position.

Efforts to improve the taste and decrease the rate release of MMI led to the development of l- carbetoxy-3-methylthioimidazole (carbimazole). Carbimazole, the pro-drug derivative of methimazole.

Both PTU and MMI are concentrated several folds by the thyroid gland and inhibit the iodination and coupling reactions of TPO Taurog described thioureylenes as potent inhibitors of thyroglobulin iodination. He suggested that a thioureylene, such as propylthiouracil (PTU-SH), would irreversibly

212 inhibit TPO-catalyzed iodination of thyroglobulin when the thioureylene-to-iodide ratio was high and reversibly when the PTU-SH-to-iodide ratio was low. In the course of the iodination reaction, the thiourelen PTU-SH would be oxidized, possibly to a disulfide dimmer, such as PTU-SS-PTU. Drug oxidation is the preferred reaction, and as long as sufficient drug is present, diverting hypoiodate (Ol-) from iodination to drug oxidation. Iodination of tyrosyl residues resumes once oxidation of the drug to disulfide products occurs by either hypoiodate or an enzyme-hypoiod complex (EOI) is complete. Under these conditions thioureylenes act as competitive inhibitors by competing with tyrosyls for hypoiodate. Conversely, at high drug concentrations, the thioureylenes are only partially oxidized, and the partially oxidized intermediate can presumably inactivate TPO by covalent binding of an oxidized form of the drug to the prosthetic hem group of TPO to prevent formation of the hydrogen peroxide-TPO complex. As a result, iodination is irreversibly blocked.

Thioureylene drugs also effectively inhibit the coupling of the DlT/MIT residues on thyroglobulin to yield the T4 and T3· This effect has been related to an alteration of the conformation of thyroglobulin brought on by the binding of the thioureylene to thyroglobulin (i. e., by the formation of a compound such as TPO-S-S-PTU).

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INSULIN AND HYPOGLYCEMIC DRUGS

Diabetes is a group of the metabolic diseases, which are described by hyperglycemia, formed in the result of insulin non-adequate production and can be accompanied by insulin and its receptors interaction disorders as well. Insulin and other hypoglycemic agents of synthetic or natural origin are used for these diseases treatment.

In 1921 Banting and Best released insulin and tested on dogs and on human first time it was applied in 1922 at 14-years old boy. In the same year insulin derived and purified from the porcine and bulls registered as a remedy in Great Britain, after a year it became available in Canada and America due to ,,Eli Lilly,, pharmaceutical company. In 1960 amino acids sequence in insulin was detected, in 1963 its total synthesis was carried out. Insulin was first remedy obtained by gene engineering and registered by FDA.

Insulin biosynthesis, structure, natural insulin types

Insulin is a natural hormone synthesized by the islet B-celIs from a single-chain, 86-amino-acid polypeptide precursor, proinsulin. Proinsulin itself is synthesized in the polyribosomes of the rough endoplasmic reticulum of the B-cells from an even larger polypeptide precursor, preproinsulin. The B chain of preproinsulin is extended at the NH -terminus by at least 23 amino acids. Proinsulin then traverses the Golgi apparatus and enters the storage granules, where the conversion to insulin occurs.

The subsequent proteolytic conversion of proinsulin to insulin is accomplished by the removal of the Arg-Arg residue at positions 31 and 32 and the Arg-Lys residue at positions 64 and 65 by an endopeptidase that resembles trypsin in its specificity and a thiol-activated carboxypeptidase B-like enzyme.

The actions of these proteolytic enzymes on proinsulin result in the formation of equimolar quantities of insulin and the connecting C-peptide. The resulting insulin molecule consists of chains A and B, with 21 and 30 amino acid residues, respectively. The chains are connected by two disulfide linkages, with an additional disulfide linkage within chain A.

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The high bioactivity of insulin depends on the integrity of the overall conformation. The biologically active form of the hormone is thought to be the monomer. The receptor-binding region consists of A-I Gly, A-4 Glu, A-5 Gln, A-19 Tyr, A-21 Asn, B-12 Val, B-16 Tyr, B-24 Phe, and B-26 Tyr. The three-dimensional crystal structure appears to be conserved in solution and during its receptor interaction.

The amino acid sequence of insulin from various animal species has been examined. It is apparent from the analysis that frequent changes in sequence occur within the inter chain disulfide ring (positions 8, 9, and 10). The hormonal sequence for porcine insulin is the closest to that of humans, differing only by the substitution of an alanine residue at the COOH-terminus of the B chain. Porcine insulin, therefore, is a good starting material for the synthesis of human insulin.

Insulin composes 1% of pancreatic tissue, and secretor protein granules contain about 10% insulin. These granules fuse with the cell membrane with simultaneous liberation of equimolar amounts of insulin and the C-peptide.

Insulin enters the portal vein, and about 50% is removed in its first passage through the liver. The plasma half-life of insulin is approximately 4 minutes, compared with 30 minutes for the C-peptide.

Regulation of insulin secretion is affected by numerous factors, such as food, hormonal and neuronal stimuli, and ionic mechanisms. In humans, the principal substrate that stimulates the release of insulin from the islet B-cells is glucose. In addition to glucose, other substrates (e.g., amino acids, free fatty acids, and ketone bodies) also can stimulate insulin secretion directly. Secretin and ACTH can directly stimulate insulin secretion. Glucagon and other related peptides can increase the secretion of insulin, whereas somatostatin inhibits its secretion.

Autonomic neuronal mechanisms also play an important role in regulating insulin release. In the sympathetic nervous system, α-adrenergic agonists inhibit insulin release, whereas β-adrenergic agonists stimulate the release of insulin. In the parasympathetic nervous system, cholinomimetic drugs stimulate insulin release.

Insulin was the first protein for which a complete amino acid sequence was determined with followed synthesis of A and B chains of human, bovine, and sheep insulin. The A and B chains were combined to form insulin in 60 to 80% yields, with a specific activity comparable to that of the natural hormone. Later was total selectively synthesized final human insulin molecule appropriately cross-linked by disulfide (-S-S-) groups in yields ranging between 40 and 50%, whereas earlier synthetic methods involved random combination of separately prepared A and B chains of the molecule.

rDNA technology has been applied successfully in the production of human insulin. Human insulin is produced in genetically engineered Escherichia coli. There are two available methods of applying rDNA technology in the production of human insulin. The earlier method involved insertion of genes, for production of either the A or the B chain of the insulin molecule into a special strain of E. coli (K12) and subsequently combining the two chains chemically to produce an insulin that is structurally and chemically identical with pancreatic human insulin. The second, and more recent, method involves the insertion of genes for the entire proinsulin molecule into special E. coli cells that are then grown in fermentation process. The connecting C-peptide is then enzymatically

215 cleaved from proinsulin to produce human insulin. Human insulin produced by rDNA technology is less antigenic than that from animal sources.

Partial syntheses and molecular modifications have been developed as the basis for SAR studies. Such studies have shown that:

1. Amino acid units cannot be removed from the insulin peptide chain A without significant loss of hormonal activity. 2. Several amino acids of chain B, however, are not considered essential for activity. Up to the first six and the last three amino acid units can be removed without significant decrease inactivity. 3. Two insulin analogues, which differ from the parent hormone in that the NH2- terminus of chain A (A1) glycine has been replaced by L- and d-alanine, respectively, have been synthesized for SAR studies. The relative potencies of the L and D analogues reveal interesting SARs. The L- and d-alanine analogues are 9.4 and 95%, respectively, as potent as insulin in glucose oxidation. The relative binding affinity to isolated fat cells is reported to be approximately 10% for the L and 100% for the D analogue.

4. Apparently, substitution on the carbon of A1 glycine of insulin with a methyl in a particular configuration interferes with the binding; hence, the resulting analogue (that of L-alanine) is much less active. Methyl substitution in the opposite configuration affects neither the binding nor the bioactivity. 5. Molecular modifications of insulin on the amino groups appear to reduce bioactivity, but modifications of the c-amino group of lysine number 29 on chain B (B-29) may yield active analogues. Synthesized N- c-(+)-biotinyl insulin, which was equipotent with natural insulin. Complexes of this biotinyl-insulin derivative with avidin also were prepared and evaluated biologically these complexes showed a potency decrease to 5% of that of insulin. Such complexes conjugated with ferritin are expected to be useful in the development of electron microscope stains of insulin receptors.

6. Alteration in the tertiary structure of insulin appears to drastically reduce biological activity as well as receptor binding. The insulin monomer has an exposed hydrophobic face that is thought to be involved directly in interacting with the receptor. Thus, loss of biological activity in insulin derivatives, produced by chemical modification, can be interpreted in terms of adversely affecting this hydrophobic region. Also, species variation in this hydrophobic region is very unusual. 216

Insulin is inactivated in vivo by: 1. disulfide bonds reduction by the glutathione insulin transhydrogenase (insulinase) in liver 2. pepsin and chymotrypsin hydrolyze some peptide bonds that lead to inactivation. 3. an immunochemical system in the blood of insulin-treated patients. The insulin receptor is a glycoprotein complex. The receptor is thought to consist of four subunits: two identical units (α and β) joined together by disulfide bonds. The α subunits are primarily responsible for binding insulin to its receptor, and the β subunits are thought to possess intrinsic protein kinase activity that is stimulated by insulin. The primary effect of insulin may be a kinase stimulation leading to phosphorylation of the receptor as well as other intracellular proteins. Additionally, insulin binding to its receptors may result in the generation of a soluble intracellular second messenger (possibly a peptide) that may mediate some insulin activity relating to activation of enzymes such as pyruvate dehydrogenase and glycogen synthetase. The insulin-receptor complex becomes internalized and may serve as a vehicle for translocating insulin to the lysosomes, in which it may be broken down and recycled back to the plasma membrane. The half-life of insulin is about 10 hours. No competitive antagonists or partial agonists of insulin exist yet. Usually, exogenous insulin is weakly antigenic. Insulin antibodies have been observed to neutralize the hypoglycemic effect of injected insulin. The antibody-binding sites on insulin are quite different from the sites involved in binding of insulin with its receptors. The binding of insulin to its target tissue is determined by several factors. The number of receptors in the target issue and their affinity for insulin are two important determinants. These factors vary substantially from tissue to tissue. Another important consideration is the concentration of insulin itself. Elevated levels of circulating insulin decrease the number of insulin receptors on target cell surfaces and vice versa. Other factors that affect insulin binding to its receptors include pH, temperature, membrane lipid composition, and ionic strength. It is conceivable, therefore, that conditions associated with insulin resistance, such as obesity and type I and type II diabetes mellitus, could be caused by altered receptor kinase activity or impaired generation of second messengers (low-M, peptides), increased degradation of the messenger, or fewer substrates (enzymes involved in metabolic activity) for the messenger or receptor kinase. Insulin preparations. Insulin shows its biological activity only in monomer form. In a solution it is unstable, which is due to its physicochemical and chemical properties. The main change to which it undergoes is its dimeration due to hydrogen bonds between B26 and B24 amino acids residues. Insulin dimmer shows very high antigenic activity and 30% of allergic reactions develop in dimmer existence. Beside this insulin can undergo chemical alteration in a solution. If insulin is stored at 4° C at pH 2-5, deamidation of the asparagine at A21 occurs. C terminal asparagine, under acidic conditions, undergoes cyclization to the anhydride which in turn may react with water leading to deamidation. The anhydride may also react with the N-terminal Phe of another chain to yield a crosslinked molecule. If stored at 25° C the inactive deamidated derivative constitutes 90% of the total protein after six months. If stored at neutral pH, entirely 217 different reactions occur. Here the deamidation occurs on the Asn residue at B3, and the products, the aspartate and isoaspartate containing insulins, are equiactive with native insulin. More deleterious transformations are also possible, including chain cleavage between Thr (A8) and Ser (A9) and covalent cross linking, either to another insulin chain or to protamine, if present. These processes are relatively slow compared to the deamidations, but have the potential to cause allergic reactions.

Insulin deamidation in acidic environment

Insulin deamidation in neutral environment

For getting insulin stable preparations B chain amino acid content is changed its hexamer is obtained connected with 2 atom zinc (in the organism insulin also stores like this). For increasing action duration, its suspension with protamin is obtained. Insulin preparations are: Regular Insulin - crystalline zinc insulin; Crystalline (uncomplexed) insulin may be given intravenously.

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Lispro insulin (Humalog®) - structurally modified human recombinant insulin: change eliminates the ability to dimerize, results in faster absorption rates.

Insulin Aspart (Novolog®) - structurally modified human recombinant insulin, change eliminates the ability to dimerize/hexamerize, results in faster absorption rates - similar to Lispro.

Glulisine Insulin (Apidra®) - structurally modified recombinant human insulin, change eliminates the ability of Glulisine insulin, to dimerize or form zinc hexamers, - results in faster absorption rates - similar to modified insulins

Inhaled Insulin (Exubera®) - rapid acting NPH Insulin – intermediate - Neutral Protamine insulin treated with protamine and zinc @ neutral pH (7.2), - protamine is a basic protein that readily complexes with insulin and zinc to yield particles that slowly dissolve in body fluids Lente insulin - insulin zinc suspension - consists of a mixture of two forms of insulin zinc suspension: 1. amorphous form - dissolves rapidly 2 crystalline form - less soluble, slowly absorbed, and similar in duration to NPH insulin i.e., intermediate-acting. Ultralente insulin - first long-acting prep, crystalline insulin zinc particles - large crystals, slow absorption, used to provide a basal level of insulin. 219

Glargine Insulin (Lantus®) - pH 4 solutions, - A substituted form of insulin in which Asn at position 21 is replaced by Gly and two Arg residues are added to the C-terminus of the B- chain, this insulin analog has low solubility at neutral pH, results in slow release over 24 h with no pronounced peak, can be used as basal insulin injection on a once daily injection basis Detemir Insulin (Levimir®) fatty acid derivatized, long-acting, fatty-acid moiety is attached to Lys-29 that is now the last amino acid of the B chain, lipid moiety responsible for slow absorption in subcutaneous space. Once in the circulation, detemir is bound to albumin, slowing its transport across the endothelium. Early data suggest the “weight neutrality effect” may be due to FA, enabling more efficient crossing of BBB, enhancing insulin’s appetite regulatory effect

ORAL HYPOGLYCEMIC AGENTS

According to the chemical structure internally used hypoglycemic drugs are divided into:

- Sulfonylurea derivatives (SUD) - amino acids derivatives-meglytinides - biguanides - thiazolidiones - alfa-glycozidase inhibitors

Sulfanyl urea derivatives (SUD)

Preparations of this group are:

I generation - Butamide (Tolbutamide), Tolazamide, Acetohexamide, Chlorpropamide

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Tolbutamide Acetohexamide

Chlorpopamide Tolazamide

II generation - Gliburide /Glibenclamide/ (Maninil), Glipizide, Gliclazide /Diabeton/

Gliburide Glipizide

Gliclazide

III generation - Glimepiride (Amaril)

Glimepiride

The classic first- and second-generation sulfonylureas share many attributes; the newer third generation materials notably different. The principal effect of these drugs isto stimulates the release of insulin; An aditional effect of sulfonylureas is suppression of gluconeogenesis in the liver. Sulfonylureas are weak acids due to the marked delocalization of the nitrogen lone electron pair by the sulfonyl group. Their pKa's cluster around 5.0 and they, like other weak acids, are strongly protein bound. As such, sulfonylureas compete with other weak acids for protein binding sites, which may result in elevated levels of free drug in the presence of other protein binding drugs.

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Metabolism. Metabolism plays an important role in the biological properties of sulfonylureas. Tolbutamide is metabolized in the liver to p-hydroxy tolbutamide. Although this metabolite retains about 35% of the activity of the parent compound; it is converted very rapidly to the inactive tolbutamide 4-carboxylic acid, so that tolbutamide is the least potent of the sulfonylureas. Tolazamide is also oxidized to a carboxylic acid by the' same two-stage process, and can in addition yield 4'-hydroxy tolazamide. These hydroxylated materials are less potent than tolazamide, but more so than tolbutamide. Consequently, tolazamide exhibits both increased potency and increased duration of action when compared with tolbutamide. The acetyl carbonyl group of acetohexamide is rapidly reduced to yield 4-(l- hydroxyethyl) acetohexamide, which has 2.5 times the hypoglycemic activity of acetohexamide and also accounts for the long duration of action of acetohexamide. Acetohexamide can also afford a small amount of 4'-hydroxyacetohexamide, an inactive metabolite. Chlorpropamide undergoes relatively slow hydroxylation on the propyl chain to afford 2'- and 3'-hydroxy-chlorpropamide. Because these processes are slow, chlorpropamide is a long-lasting drug.

The second-generation agents’ glyburide and glipizide undergo similar and interesting biotransformations. Glyburide affords trans4'-hydroxyglyburide as the major product, accompanied by some cis-3'-hydroxy material. The 4'-hydroxy metabolite retains about 15% of the activity of the parent compound. Clipizide undergoes the same transformations. In addition, it undergoes an interesting cleavage of the pyrazine ring to afford the N-acetyl derivative shown. None of these metabolites exhibits useful therapeutic activity.

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Structure-activity Relationships 1. There must be reasonable bulk on the urea nitrogen; 2. Methyl and ethyl compounds are not active. 3. Usually, there is only one (normally substituent para) on the sulfonylaromatic ring. 4. Many simple substituents are active, and the (p-arylcarboxamidoethyl) grouping seen in second generation compounds is consistent with high potency. 5. Among these compounds, it is thought that the spatial relationship between the amide nitrogen of the substituent and the sulfonamide nitrogen is important.

Third-generation Sulfonamides Glimepiride. Glimepiride, a sulfonylurea with a quick onset of action and a long duration of action, may bind to a different protein in the putative sulfonylurea receptor than earlier drugs, and may exert its hypoglycemic effect with less secretion of insulin, unusual of the earlier sulfonamides. It has also been suggested that glimepiride may cause translocation of the GLUT-4 glucose transporter from the cytoplasm to an active position in the cell membrane. Thus, while all sulfonylureas exhibit both insulin-secreting and extrapancreatic activities, glimepiride relies upon extrapancreatic effects for a greater portion of its hypoglycemic effect. Perhaps for this reason it is said to be less likely to produce unwanted hypoglycemia. Glimepiride is metabolized, primarily in liver, The M-1 metabolite appears to be formed by Cytochrome P-450 enzymes (mostly CYP-2C9) in liver microsomes. Further metabolism catalyzed bycytosolic dehydrogenases affords the carboxylic acid metabolite M- 2. Metabolite M-1 exhibits significant hypoglycemic activity in man, whereas metabolite M- 2is inactive.

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Meglytinides Repaglinide Repaglinide, a nonsulfonylurea (but still an acidic molecule). This drug is even more rapid- and short acting than other hypoglycemic drugs. It is beyond doubt that replaglinide is at least 5 times more potent than glyburide on intravenous administration; the difference on oral administration is about 10-fold.

In an interesting conformational study, it was shown that repaglinide and several other active nonsulfonylurea hypoglycemics, as well as the sulfonylureas glyburide and glimepiride, displayed a comparable U-shaped conformation when analyzed by molecular modeling. In this conformation, hydrophobic cycles were placed at the end of each branch and a peptidic bond was at the bottom of the U. Several inactive analogues of repaglinide and the poorly active drug meglitinide displayed a different conformation, with a greater distance between the hydrophobic cycles. Biguanides /guanidine derivatives/

Galegine /Galega officinalis plant/ Several guanidine derivatives are active as antihyperglycemic agents, of which metformin and phenformin are of most interest. Shortly thereafter, it was found to produce increased serum lactic acid levels, which sometimes progressed to lactic acidosis and was associated with 50% mortality.

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The mechanism of action of metformin:  It is sometimes classified as an inhibitor of hepatic glucose production and the drug does not induce hypoglycemia at any reasonable dose. For that reason, metformin is usually said to be an antihyperglycemic (or euglycemic) rather than a hypoglycemic agent.  Overall, the drug appears to increase glucose utilization.  Inhibition of gluconeogenesis appears to be an important component of the drug's activity,  Effects on glucose transporter proteins have also been suggested.

Thiazolidinediones The thiazolidinediones also known as the "glitazones," are sometimes referred to as insulin enhancers. They are exemplified by ciglitazone, the first of the glitazones.

The glitazones lower blood glucose concentrations by improving sensitivity to insulin in target tissue, which includes adipose tissue, skeletal muscle and liver. These agents are dependent upon insulin for their activity. The drugs appear to enhance insulin action, especially in liver, muscle and fat tissue where insulin-dependent glucose transport is essential. An interesting effect of these antihyperglycemic drugs is on the peroxisome- proliferator activated receptor: PPARy. These drugs act as agonists upon binding to PPARy, which preferentially binds to DNA activating transcription of a wide variety of metabolic regulators. The regulators increase expression of a number of genes involved in the regulation of glucose and lipid metabolism. The thiazolidinediones differ by the nature of the groups attached to the 2,4- thiazolidinedione nucleus. These agents are extensively metabolized with all metabolic changes occurring on or adjacent to the aryl group found in the side chain. Considerable interest in their metabolism since the hepatic toxicity may be associated with a metabolite.

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α-Glucosidase Inhibitors To be absorbed from the gastrointestinal tract into the bloodstream, the complex carbohydrates we ingest aspart of our diet, primarily starch and sucrose, must first be hydrolyzed to monosaccharides. The rationale for the exglucosidase inhibitor class of drugs is that bypreventing the hydrolysis of carbohydrates their absorption could be reduced. Starch is normally digested by salivary and pancreatic ex-amylases to yield disaccharides (maltose), trisaccharides (maltotriose), and oligosaccharides (dextrin). The oligosaccharidases responsible for final hydrolysis of these materials are all located in the brush border of the small intestine, and consist of two classes. The β-galactosidases hydrolyze β-disaccharides such as lactose, whereas the α-glucosidases act on ex-sugars such as maltose, isomaltose, and sucrose.

An extensive search for ex-glucosidase inhibitors from microbial cultures led to the isolation of acarbose from an actinomycete.

Acarbose Extensive structure-activity investigations revealed that active inhibitors had a common active site comprising a substituted cyclohexane ring and a4,6-dideoxy4-amino-D- glucoseunit known as carvosine. It appears the secondary amino group of this core structure prevents an essential carboxylgroup of the α- glucosidase from protonating the glycosidic oxygen bonds of the substrate. More recently, screening programs of small molecules have yielded several other α- glucosidase inhibitors, of which miglitol has been introduced to the market. Most of the substances tested resemble simple amino sugars.

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