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Lecture 20

CHEM669 Lecture 20: project

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University of Alberta
Fredrick West

Page 1 of 18 Introduction Neuropathic pain that affecting millions of people around the world is the result of any organ’s damage or dysfunction in the nervous system and they are classified into central and peripheral pains. The painful diabetic neuropathy (PDN) and post herpetic neuralgia (PHN) are the most common types of Peripheral neuropathic pain. PHN comes from a herpes zoster infection whereas PDN comes from the long-term complication of diabetes. The central neuropathic pain mechanism are not fully understood. Currently there is some effective treatments for neuropathic pain but only 40 to 60% of patients after could experience pain relief. So these available treatment are usefulness in some patients. Several trials assessed the health status, pain relief, tolerability and quality of life in patients with central neuropathic pain after treatment with pregabalin (PGB) and fortunately, it showed potent ability for treatment of 1,2 these patients. 3-Isobutylgaba or 3-aminomethyl-5-methyl-hexanoic acid has 2 isomeric conformations, R and S, which only its S enantiomer or PGB (S-(+)-3-isobutylgaba) is pharmacologically active and its substituent at the 3-position simplify its penetrate across the blood–brain barrier. Although PGB ( Lyrica) is a lipophilic analogue of GABA(inhibitory transmitter gamma amino butyric acid), PGB doesn’t bind to GABAA or GABAB receptors and doesn’t interact with other GABA uptake transporter drugs. So, its mechanism of action is completely new. Activity of PGB and gabapentin (GBP or Neurontin) are related to an α -δ2binding site in voltage-gated calcium channels in the central nervous system. 3,4,These channels are important member of neurons, so any disturbances in them cause several neurological diseases; for example, epilepsy, muscle weakness, and migraine. A genetic illness that is known as Spinocerebellar ataxia type 6 has some effects on the P/Q-type voltage- dependent calcium channel (P/QCC) regulation. GBP and PGB can counteract the channel deregulation found in this transgenic disease.6 So, PGB is used as a potent drug for treatment of analgesics, anxiety,and epilepsy. Also it is used for other therapy such as inflammation, gastrointestinal disorders, insomnia, alcoholism and different psychiatric problems like mania and bipolar disorders. Recently PGB was approved by both USA and EU for partial seizures therapy and by only EU for anxiety disorders. Not only its structure, but also its pharmacological activity is similar to Gabapentin. 7,8 In 1994, GBP as an add-on therapy for epilepsy showed positive effects against neuropathic pain and anxiety in Human trails. Although GBP and PGB interact with the same binding site, and their pharmacologic profile is similar, PGB has superior potency and pharmacokinetics than GBP. Moreover, PGB is absorbed more rapidly . So, that’s the reason why PGB is identified as more ‘powerful’ drug and indicates better results in treatments. It has been approved for the treatment of neuropathic pain related to PDN and PHN. 4,9 Gababutin (GBB) is a five-membered ring analogues of gabapentin. It indicates a binding affinity of 420nM at the α -2 binding site. It seems that its five- membered ring cannot optimally fill the binding pocket space. In order to create compounds with better potency to gabapentin, probably attachment of some alkyl group10to GBB’s ring help it to fill the binding pocket. It has been observed that although over 20 antiepileptic drugs are developed for epilepsy, these drugs are effective only in 30% of epilepsy patients for unknown reasons. P- glycoprotein is responsible for exporting these drugs and overexpressing them in blood. Figure1. Structure of PGB, Gabapentin, and Gababutin. 10 Page 2 of 18 P-glycoprotein, in patients who are resistant to antiepileptic drugs, is expressed in blood at higher levels compared to drug-responsive patients. Several antiepileptic drugs are p-glycoprotein’s substrates or inhibitors such as phenytoin, Topiramate, Lamotrigine, Levetiracetam, Eslicarbazepineacetate, Phenobarbital, S-licarbazepine, Oxcarbazepine, carbamazepine. Recently synthesized epilepsy drugs such as zonisamide (ZNS), pregabalin (PGB), and rufinamide (RFM) are not P-glycoprotein substrate and their mechanism is different. Five classes of drugs have effective results on Generalized anxiety 12 disorder (GAD) disease which PGB belongs to the α -δ li2and class. PGB also often prescribed off- label for some clinical therapies such as alcohol/narcotic withdrawal, attention-deficit disorder, trigeminal neuralgia, and restless legs. Side effects of SSRI and SNRI antidepressant drugs such as nausea, dizziness, diarrhea, and sexual dysfunction is attributed to their activity at receptors not directly related to their mechanism12However, a binding assay indicate that PGB selectively binds to its receptor and has not any side effects. Pregabalin’s Target and Mechanism Voltage-dependent calcium channels (VDCCs) play an important role in neuron cells. Appearance of some neurological diseases is for the sake of disturbances in VDCCs. VDCCs are divided to 2 categories, P-type VDCCs and Q-type VDCCs which are predominant in Purkinje cells and in rat cerebellar granule cells, respectively. VDCCs as heteromeric complexes mediate calcium penetration into cells and this penetration depends on changes in membrane potential and depolarization of axonal membrane. It means that VDCCs are closed at resting membrane potential. In fact, these entered-cell calcium ions acts as a second messenger for sending electrical signals. Releasing and propagation of neurotransmitters requires a vesicle to bind with presynaptic membrane and all of these happens in the presence of Ca . When the PGB binds to α -δ proteins, it reduce the Ca +2 availability required for 2 5,6,12,13 membrane fusion and promote release of neurotransmitters. VDCCs contain several subunits, α ,1 α2-δ, β, and γ which only α -δ2acts as a target for PGB. Subsequently, there are four different α -δ 2 proteins in mammals which each of them coded by a different gene. Channel proteins Related gene Targeting Drug α2-δ type 1 (α 2δ1) CACNA2D1 PGB and GBP α2-δ type 2 (α 2δ2) CACNA2D2 PGB and GBP α2-δ type 3 (α 2δ3) CACNA2D3 No Drug α2-δ type 4 (α2-δ4) CACNA2D4 No Drug Each of these protein contains 997—1150 amino acid residues. Although α -δ is2a single polypeptide protein, it will cleaved into two polypeptides: α and δ. The δ portion retains in the cell membrane because it contains so much carboxyl terminal residues. However, the α 2 portion retains in extracellular. The α2and δ portion are bound together by disulfide bonds (Figure 2). Evaluation of Arginine 217 (R 217) in α -δ 2 helps researchers understand the mechanism of PGB more clear. The triple arginine is available only in α2-δ1 and α2-δ2. Figure 2. α 2δ structure. RRR is the α 2δ1 and α 2δ2 14 sequence while RNR is the α -23 and α -δ2 sequence. Page 3 of 18 Mutagenesis of the central arginine in both α -δ1 and2α -δ2 has n2t any impact on PGB binding. Scientist evaluated the effect of mutation of R 217 in α -δ1 an2 R 279 in α -δ2 to Ala2ine on PGB mechanism. Results of this evaluation proved that mutated R 279 has not any influence on PGB efficacy, nevertheless, mutated R217 will blocked PGB activity in all assays. Therefore, the PGB therapeutic activity is related to α -δ12subunit binding. 14,15 Before 1996, it had been known that voltage-gated sodium channels, GABA reuptake transporters, and GABAA receptors are the only binding sites for treating the epilepsy. After the identification of the α -δ s2bunit proteins as the high affinity binding site for PGB and GBP in 1996, scientist can increase the effective results in treatment 6,14 of epilepsy. A 3H-gabapentin binding assay indicated that GABA has not any displacement in 3H-gabapentin binding site and the R(-) enantiomer of PGB with an IC50=600nM, compared to its S(+) enantiomer with IC50=37nM, was much less effective related to this displacement. This study also confirmed that PGB is more effective than GBP. Since this study indicated that the purified receptor’s N-terminal sequence was same as the sequence of α -δ subun2t in VDCC, binding of PGB to α -δ subunit was 2 14 confirmed. Displacement binding studies indicated that PGB binds selectively to the α -δ1 and2α -δ2 subun2t in the brain. By decreasing calcium penetration in presynaptic terminal in brain, depolarization and subsequentl7,15 decrease in neurotransmitter release happens. It can also suppress muscles neuronal responses. The α -2 site acts as an auxiliary protein in VDCCs. When PGB binds the α -δ subunit, 2 calcium penetration at nerve terminals will be decreased. Consequently, the release of several neurotransmitters such as glutamate, noradrenaline, and substance will decrease, too. Since PGB even at high concentrations doesn’t completely block VDCCs or transmitter release, the safety implications of this drug even in overdose cases is remarkable. 5,16,17 Figure 3. A)Structures of calcium channel subunits. B) a three-dimensional structural model of 8 human’s α -δ2 protein. ( Taylor, C. P.; Angelotti, T.; Fauman, E. Epilepsy Research 2007, 73 (2), 137) AS it is shown in the figure 3(A), each VDCC contains four homologous domains. In α -δ binding si2e, 2+ Ca is entered the cell through a pore which is formed by α transmelbrane subunit. The δ domain and α2subunit are fused to each other by disulfide bond, while the intracellular β subunit modulates the functioning of α -2 proteins. The γ subunit is an embedded glycoprotein inside the cell membrane. The 3-postioned bulky aliphatic substituent on the GABA backbone in PGB cause to bind to α -δ binding 2 sites instead of GABA receptors. Figure 3(B) shows a three-dimensional structural model of human’s α2-δ1 protein. Red and yellow arrows are alpha helices and beta sheets, respectively, which 2+ representing the amino acid sequence order. The purple ball is a Mg ion which is tied to some amino acids such as D (Asp), S (Ser), and T (Thr). Page 4 of 18 R217 (Arginine) is the residue which is responsible for high-affinity binding between PGB and extracellular domain of α -δ1 subunit.,12,18 2 Hit-to-lead Over the past10–20 years, because of new signaling pathway developments, only small number of new drugs has been identified. In the mid-1990s, researchers started to work on new strategies to find a drugable hit and calcium channel α 2δ subunits fell into this category. At first scientist did not know any known drugs for α -δ 2 subunits, also there were no known homologous proteins as drug targets. Binding of GBP to the α -δ2 proteins of VDCCs changed everything. They found out that PGB can bound to α -21 proteins, too. It indicated that2α -δ is a drugable target, and can open up a new strategies for treating epilepsy, pain, and anxiety. Although in 1912, Phenobarbital used as a sedative hypnotic and antiepileptic drug, it took around 50 years for understanding its mechanism. Figure 4. Drugs that are studied for synthesizing PGB. In the 1920s, Pentylenetetrazole was used as a convulsant drug although its usage was not standardized, and since it wasn’t reproducible, its usage was problematic too. In1920s, after introduction of Phenytoin, a breakthrough in developing new anticonvulsant drugs happened. So, between 1930s and 1960s, several antiepileptic drugs such as Phenytoin, Chlordiazepoxide, and Diazepam are introduced to the market. Because of their unclear mechanism of action in animal models, they had to develop. In the 1970s, they understood that diazepam shows the characteristics of an allosteric modulator and can increase GABA’s ability to block pre-synaptic activity. Bicuculline indicated that can inhibit ion penetration through the GABAA receptor. In1950s, researchers proved that phenobarbital also like the benzodiapine drug had an allosteric impact on the GABAA receptor. In 1962, a lipophilic derivative of GABA, Baclofen, was synthesized and in 1972, it entered the market for spasticity treatment. Baclofen, barbiturates and Diazepam interact with both GABAA, and GABAB receptors. In 1980s, some attempts have also been made for developing other anticonvulsant drugs such as Vigabatrin and Tiagabine that can affect synaptic cleft. Tiagabineenters the brain and binds to the GABA transporter GAT-1 to treat partial epilepsy. Vigabatrin irreversibly attach to GABA aminotransferase and decreased the degradation of GABA. In 1974, Gabapentin (GBP) as an analog of GABA is synthesized. Its lipophilicity cause entering the brain easily, and it was non-hydrolysable too. The maximal electroshock model of epilepsy for GBP, in 1995, indicates that GBP can block induced seizures. Because of resemblance in structure of GABA and GBP, scientists tried to find any relation between GABA receptors and GBP. A GABA reuptake inhibitor like Nipecotic acid can induce a neonatal optic nerve depolarization. Incubating GBP with Page 5 of 18 human neocortical slices will increase GABA levels slightly. Although the brain GABA level measurement by 1HNMR in epilepsy patients who are treated with GBP proves this small increase, animal studies indicate no changes in GABA levels. So, they concluded that the drug’s mechanism is not related to human GABA levels. Some studies also showed that GBP hasn’t any impact on extracellular GABA levels. Although in vitro data of GBP indicates that it can inhibit GABA transaminase, block amino-acid transferase, and stimulate glutamate decarboxylase, in vivo data of GBP cannot confirm them. In 2001, a new group showed that because GBP can inhibit calcium channels or induce potassium currents, it can use as an anticonvulsant, too. Moreover, since GBP was inactive in some assays including GABA binding to recombinant receptors and G protein activation measurement in mammalian cells, anticonvulsant effect of this drug is not related to GABA receptor. At Northwestern University in 1991, PGB was synthesized by the Silverman and his group who developed several 3-alkyl derivatives of GABA which are active against the transferase enzyme. The maximal electroshock assay indicated that some of these derivatives are anticonvulsant and the (S)-3- isobutyl analog (PGB) was the most active molecule in seizure treating. Since PGB, GBP, and GABA are similar in structure, at first it was thought that all of them has the same mechanism. Using a binding assay for 3H-gabapentin on animal model indicated that GABA and baclofen didn’t show any binding to the receptor in the brain, however, GBP showed binding and PGB with high potency, bound to the receptor, too. Additionally, data indicate that PGB and GBP are active anticonvulsant drug which both are active in α 2δ binding site, not GABA receptor. 8,11,14 Properties and SAR of Pregabalin PGB with the Molecular Formula of C8H17NO2 and Molecular Weight of 159.23 is thermally stable and insoluble in cell membranes and lipids because of its hydrophilic structure and its zwitterionic structure. With the help of specialized transporter system (system L) it can cross gut membrane barriers, cell membranes, and blood-brain barrier. Its entry into the brain via system L has not any effect directly on its pharmacological actions. PGB pharmacological activity and its high-affinity binding to α2-δ subunits of VDCCs depends on its 3-positioned aliphatic side chain. Moreover, its side chain allow system L to transport it and decrease PGB activity at GABA receptors. 8,19 The Suggested dose for PGB is 75mg (twice per day) or 50mg (three times per doy). PGB solution is stable around 8 hours at +4 C with a recovery of 98.0%. Recovery of PGB in freeze-thaw study is 99.00%. 20,21Although PGB currently approved as a hard crystallinecapsule for oral use, its new oral formulation is a clear, colorless, and flavored solution. Sucralose and artificial strawberry are added as a sweetening agent and flavor, respectively, to mask PGB bitter taste. More than 90% of PGB absorbed rapidly, also its administration with food doesn’t change its bioavailab22ity. Its biological half- life is around 6.3-11.5 hours. Figure 5. SAR study of PGB. 19 For structure-activity relationships (SAR) investigation of PGB, a number of synthesized alkylated analogues were assessed in vitro for binding α -δ subunits. Addition of alkyl groups on PGB scaffold 2 can clarify SAR and biological activity. Ability of all compound for displacement of 3H-gabapentin from brain tissue was tested. The antiepileptic activities of compounds were evaluated in vivo and then the result is expressed as an increase in paw withdrawal latency in rats. The following table will compare the activity of some analog compounds with PGB. Page 6 of 18 NO. Structure Affinity for Affinity for system L α2-δ transporter Substitution of isopropyl 15 Fold No change group with the diethyl decrease 1-Ethylpropyl analogue 2.5 Fold No change weaker Transposition of the Decrease Decrease isobutyl group to the C2 position Substitution of a methyl Complete loss Decrease group R to the carboxylic of affinity acid Substituents of methyl 40 to 100-fold 3-5-Fold increase and ethyl at the C-3 decrease position Substitutions at the 2-Fold Sharp decrease positions α and β to the increase carboxylic acid Methyl at α position of 2-Fold Complete loss of amine in a syn increase affinity orientation Methyl group in the 4-S 20-Fold 5-Fold increase orientation decrease methyl group in the 4-R 4-Fold No change orientation increase According to the Figure 5 and the above table, it is now clear that substitution of different group on specific positions of PGB (C2, C3, C4, and Cα of amino side chain) can change its affinity. Page 7 of 18 Med. chem. Synthesis of PGB There are a few different routes for synthesis of PGB. Through the following steps and by using 3-isobutylglutaric acid and acetic anhydride as starting materials, we can synthesize PGB. Since impurities have a significant effect on the quality and safety of the drug, manufacturers try to minimize the percent of impurities. Six different impurities that have been identified during the development process of PGB such as 4-isobutylpyrrolidin-2-one, Isobutylglutaric acid , (R)-(-)-3-carbamoylmethyl Scheme 1. Synthesis of PGB. 24 -5-methylhexanoic acid, (R)-(-)-3-aminomethyl -5-methylhexanoic acid, (S)-3-aminomethyl-5-methylhexanoic acid isobutyl ester, and (S)-3- aminomethyl-5-methylhexanoic acid isopropyl ester. The following reactions show how these impurities appear.24 Scheme 2. Impurities that will be produced through this synthesis. Page 8 of 18 There is another high yield strategy for PGB synthesis in which chiral squaramide organo-catalysts such as C1, C2, C3, and C4 are used to catalyzed the Michael addition reaction between Meldrum acid and an aliphatic nitroalken25 Using 5mol% C3 catalyst will produce PGB with the highest enantiomer selectivity and high yield. 25 Scheme 3. Synthesis of PGB and Chiral squaramide organo-catalysts. By using Jacobsen’s HKR strategy, PGB can be produced with simple high enantioselective procedures. Highly availability of starting materials and catalyst for these synthesis is one o26 its advantages. Scheme 4. Synthesis of PGB and Chiral squaramid26 organo- catalyst. Page 9 of 18 In scheme 5, PGB has been synthesized through the formation of hemiaminal intermediate. If we replace ammonia with chiral amines (S-alpha methyl benzylamine or S-phenylglycinol), asymmetric PGB will be produced. 27 27 Scheme 5. One of the important route for synthesize of PGB which is not used in scale up. Preclinical Animal models PGB is a compound that indicates a potent activity in several anticonvulsant animal model Tests. An experiment of maximal electroshock test on some rates showed that PGB had an ED = 1.8 mg/kg. It 50 also estimated that the maximum plasma Concentration (C max) of PGB is 1.6 µg/ml. Because the plasma PGB concentration of this animal model is the range of human’s plasma concentration, it means this model is a successful clinical trials for PGB. The dosage of PGB in both pain associated and seizure models in rats is in the same range. When the concentration of PGB is millimolar, it can increase the activity of glutamic acid decarboxylase in vitro, however, reaching this concentration in vivo is impossible. Measuring the concentration of PGB during the micro-dialysis study of rat’s brain after anticonvulsant doses indicates that PGB concentrations is pretty below to be able to active the enzyme. Another study showed that PGB will never alter the brain’s GABA concentration. 8 Maximal electroshock studies on some mice also indicate that PGB prevents tonic extensor seizures. O scientists suggest that in treatment of partial and generalized tonic-clonic seizures, PGB would be effective. Another study confirmed that PGB can prevent generalized tonic-clonic seizures. 5 Also, it cannot inhibit GABA transport in vitro. So, PGB neither mimic GABA nor increase GABA action pharmacologically. In seizure and pain animal models, PGB needs a high affinity binding at α -δ 2 subunits to be active pharmacologically and can inhibit seizures or pain in vivo. PGB affinity binding at α2-δ subunits or its transportation via system L are the most important factors that will affect the result of treatment with PGB. In one animal mode on genetically modified mice with defective α -δ 2 subunits, the pain-relieving and anticonvulsant property of PGB decreased. However, the anticonvulsant and pain-relieving actions of phenytoin and morphine in these genetically modified mice did not changed. In fact, during this mutation, the R 217 residue of wild type mice was converted to A (alanine). Using PGB in these modified mice showed 20 fold decrease in binding affinity of α -δ 2 subunits in comparison to wild type mice. So, these data indicate that the accessibility and availability of α 2δ subunits has a main role in pharmacological actions of PGB. Evaluation of PGB;s Page 10 of 18 pharmacology indicated that this drug is a unique and amazing drug for treating epilepsy and chronic pain. Although anima8 models predict that PGB can inhibit partial and generalized seizures, it has a few side effects on brain. In cultured neocortical neuron studies again it has been proved that PGB has not any activity with GABA receptors, however, during treatment with Diazepam, the GABA-evoked cur
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