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- 1. The Discovery of Raltegravir (MK-The Discovery of Raltegravir (MK- 518), an Integrase Inhibitor for the518), an Integrase Inhibitor for the Treatment of HIV InfectionTreatment of HIV Infection By Dr. Michael Rowley IRBM, via Pontina Km 30,600, Pomezia, Rome 00040, Italy Summarized by Qinglin Che for Synta Chemistry Enhancement Program
- 2. Some Facts about HIV/AIDS 2.7 million in 2007 2.0 million in 2007 Total in 2007 33 million
- 3. Schematic Illustration of HIV-1 Virion Illustration of HIV-1 Virion Lipid bilayerLipid bilayer Viral envelopeViral envelope 72 spikes72 spikes Structure of Hiv particle Viral envelopeViral envelope
- 4. Image of HIV Infected T-Cells Size of HIV Particle: ~100-150 nmSize of HIV Particle: ~100-150 nm HIV-1 particles assembling at the surface of anHIV-1 particles assembling at the surface of an infected macrophageinfected macrophage
- 5. HIV Viral Life Cycle CXCR4CXCR4 CCR5CCR5 Intigrase inhibitorIntigrase inhibitor 0 until 20080 until 2008 Reverse Transcriiptase InhibitorReverse Transcriiptase Inhibitor 11 (NRTI) + 4 (NtRTI)11 (NRTI) + 4 (NtRTI) Protease inhibitorProtease inhibitor 99
- 6. The Chemistry of Integrase
- 7. HCV NS5B inhibitors Finding the lead: From HCV NS5B to HIV Integrase inhibitors HIV integrase & HCV NS5B -share mechanistic similarities - Need two divalent metal ions for their phosphoryltransferas e activities.
- 8. Compound 3: Dihydroxy pyrimidine acid Low permibility, Too polar Compound 4: HCV NS5B inactive HIV integrase IC50 85 nM Finding the lead: From HCV NS5B to HIV Integrase inhibitors HCV NS5B inhibitors
- 9. Discovery of L-870812 and L-870810Discovery of L-870812 and L-870810 Compound 5:Compound 5: •formform West PointWest Point •Improved PKImproved PK •Low potencyLow potency Compound 4:Compound 4: •FromFrom IRBMIRBM •high potencyhigh potency From compound 4 & 5, after optimization, L-870810 was discovered at West Point Con of (4): a. low activity in the cell-based assay > 5 µM b. low bioavailability (~15%) c. High plasma protein binding Compound L-870810 (MK-624):Compound L-870810 (MK-624): •formform West PointWest Point •Discontinued after Phase II due toDiscontinued after Phase II due to toxicity in dogs during long termtoxicity in dogs during long term dosingdosing
- 10. Lead Optimization: Investigation of Benzyl AmideLead Optimization: Investigation of Benzyl Amide 1. NH is needed 2. 1-2 methylene linker is required 3. Heterocyle ok, Potency correlated with Log P. 1. Branching linker is no good. 1. NH is needed 2. 1-2 methylene linker is required 3. Heterocyle ok, Potency correlated with Log P. 1. Branching linker is no good.
- 11. Lead Optimization: Investigation on Benzyl SubstitutionLead Optimization: Investigation on Benzyl Substitution Conclusion: 1. Para > meta > ortho 2. 4-F > 4-Cl > 4-Br > 4-CF3 > others Conclusion: 1. Para > meta > ortho 2. 4-F > 4-Cl > 4-Br > 4-CF3 > others
- 12. After Extensive Investigation…..After Extensive Investigation….. cell penetration, protein binding, PK and other parameterscell penetration, protein binding, PK and other parameters
- 13. Lead Optimization: 2-Substitution Remining Issue:. Still very low activity in cell-based assay! Even in low serum level assay (10% FBS). Compound 35, Spread CIC95 >5μM PhCH2
- 14. Compound 36: Adding a basic group confers cell activity New Lead Compound 36: -very good PK, high bioavailability: rat F 59%, dog F 93% -low clearance: rat 14 ml/min/kg, dog 0.5 ml/min/kg -protein binding (>99%) -Shift in a cell-based assay between low and high serum Conditions is high!. activity in 50% NHS (> 10μM)
- 15. for CRAC chemists: THE IMPORTANCE OF PROTEIN BINDING Lipophilic acid compound Lipophilicity (logD) ↑ albumin binding ↑
- 16. Shift in cell-based assays and Protein Binding Low protein binding low shift! But when <90%, shift < 4, good enough Optimum protein binding range 80%~90% Low protein binding High Clearance Need >80%
- 17. Direction 1 from compound 36: Simple Acyclic 2-Substituents 37: enzyme activity ok, But lower in FBS, due to low cell penetration 38: 1 methyl group FBS 10 times increase , and good NHS activity 39: 2 methyl groups, no chiral center, highly active both low and high serum 40, 41 ethyl or NH both detrimental 37: enzyme activity ok, But lower in FBS, due to low cell penetration 38: 1 methyl group FBS 10 times increase , and good NHS activity 39: 2 methyl groups, no chiral center, highly active both low and high serum 40, 41 ethyl or NH both detrimental
- 18. Compound 39, the First Candidate with development potential! Not CYP450 inhibitor, not CYP3A4 inducer, no oxidative metabolism Major Metabolite: 5-O-glucuronide
- 19. Direction 2 from compound 36: Simple Cyclic 2-Substituents A basic nitrogen was tolerated! 2-NH > 3-NH > 4-NH -NMe > -NH A basic nitrogen was tolerated! 2-NH > 3-NH > 4-NH -NMe > -NH
- 20. An important discovery: N-methylpyrimidines A side reaction?A side reaction?
- 21. An important discovery: N-methylpyrimidines
- 22. “it had two important improvements: The first was that the protein binding was considerably lower (a factor that we had struggled with in the dihydroxypyrimidine series) and, the second was that rat PK was improved, with better oral bioavailability and lower plasma clearance. We reasoned that it would be easier to improve potency in a series with good protein binding and PK than the converse and turned our attention to the N-Me pyrimidinone series.” “it had two important improvements: The first was that the protein binding was considerably lower (a factor that we had struggled with in the dihydroxypyrimidine series) and, the second was that rat PK was improved, with better oral bioavailability and lower plasma clearance. We reasoned that it would be easier to improve potency in a series with good protein binding and PK than the converse and turned our attention to the N-Me pyrimidinone series.”
- 23. N-METHYLPYRIMIDINES WITH CYCLIC 2-SUBSTITUENTS
- 24. N-METHYLPYRIMIDINES WITH CYCLIC 2-SUBSTITUENTS
- 25. N-METHYLPYRIMIDINES WITH CYCLIC 2-SUBSTITUENTS
- 26. Back to ACYCLIC BASIC SUBSTITUENTS on N-METHYLPYRIMIDINES
- 27. N-METHYLPYRIMIDINES WITH NON-BASIC POLOR SUBSTITUENTS
- 28. N-Me-PYRIMIDINES WITH NON-BASIC SUBSTITUENTS (cont.)
- 29. Evolution from Hit to Lead to preclinical candidate 66
- 31. Fight is not over DISCOVERY OF RALTEGRAVIR
- 32. DISCOVERY OF RALTEGRAVIR (cont.) Low permeability High Protein binding
- 33. RALTEGRAVIR , First-In-Class •QC IC50 10 nM •Spread CIC95 19 nM (10% FBS) •Spread CIC95 31 nM (50% NHS) •Protein Binding •PK F% 45% in rat, 69% in dog •Plasma clearance 1.8 rat, 11 dog •No CYP450 inhibitor •No CYP3A4 inducer •No UGT1A1 inhibitor •Resistance Profiling: excellent •Safety in human •Efficacy short term as monotherapy •long term in combination
- 34. The Synthesis of MK-518
- 35. S-1360 from GSKS-1360 from GSK -Licenced from Shionogi-Licenced from Shionogi -Diketoacid derivative-Diketoacid derivative -Low efficacy, extramely poor bioavailability-Low efficacy, extramely poor bioavailability -discontinued in Phase II-discontinued in Phase II GS-9137 (elvitegravir) from Gilead Licenced from Japan Tobacco Metabolised in part by Cyp3A4 co-doing with ritonayir (a HIV protease inhibitor also inhibit Cyp3A4) Phase III clinical trial Ongoing nowBMS compounds similar to RaltegravirBMS compounds similar to Raltegravir Discontinued in Phase IIb due To hepatotoxicities Other HIV Integrase Inhibitors
- 36. First-in-ClassFirst-in-Class What do you think?What do you think? First-in-ClassFirst-in-Class What do you think?What do you think? • Trial and error, learning curve, solid science,Trial and error, learning curve, solid science, be prepared of failure in the beginningbe prepared of failure in the beginning • Competition. Not guaranteed winner among otherCompetition. Not guaranteed winner among other big pharmasbig pharmas • Some luck is needed!Some luck is needed!
- 37. A Quote from Barbara Ehrenreich “From the point of view of the pharmaceutical industry, the AIDS problem has already been solved. After all, we already have a drug which can be sold at the incredible price of $8, 000 an annual dose, and which has the added virtue of not diminishing the market by actually curing anyone.” Ehrenreich, Barbara (from wikipedia) In clinical trials patients taking Raltegravir achieved viral loads less than 50 copies per millitre sooner than those taking similarly potent Non-nucleoside Reverse Transcriptase Inhibitors or Protease Inhibitors. This statistically significant difference in viral load reduction has caused some HIV researchers to begin questioning long held paradigms about HIV viral dynamics and decay. Research into Raltegravir's ability to affect latent viral reservoirs and possibly aid in the eradication of HIV is currently ongoing. Ongoing Clinical Trials: Depletion of Latent HIV in CD4 Cells
- 39. How difficult to get a first-in-class drug to the market from scratch
- 40. Raltegravir Potassium Salt: at a glance
Notas del editor
- Here is some basic statistics data upto 2002. There are totally over 42 million HIV infections in the world, mostly distributed in Africa, and in US, more than 1 million.
- Fig. 5-36 Schematic illustration of the human immunodeficiency virus (HIV)-1 virion. The viral particle is covered by a lipid bilayer derived from the host cell and studded with viral glycoproteins gp41 and gp120. HIV particles surround themselves with a coat of fatty material known as the viral envelope (or membrane). Projecting from this are around 72 little spikes, which are formed from the proteins gp120 and gp41. Just below the viral envelope is a layer called the matrix, which is made from the protein p17. The viral core (or capsid) is usually bullet-shaped and is made from the protein p24. Inside the core are three enzymes required for HIV replication called reverse transcriptase, integrase and protease. Also held within the core is HIV&apos;s genetic material, which consists of two identical strands of RNA.
- Then how are HIV infected? How HIV virus invade human cell? Basically there are five steps Binding to cd4 or other cell surface proteins, allow the virus to fuse with the cell. The lipid membrane incorporated into the cell’s membrane, while the viral core enters the host cell Under the help of reverse transcriptase, the virus copy the HIV RNA to cDNA. High error rate, caused quick evolution. The double strand DNA then enter the nucleus with some protein including integrase, which spice the virus DNA in the the host cell DNA, forming HIV provirus. The synthesis of viral genome begin with transcription of DNA to RNA, and from RNA to protein. RNA and proteins assemble at the membrane and bud off the host cell.
- HIV-1: I) donor DNA; II) integrase-catalyzed 3&apos; processing; III) integrase-catalyzed strand transfer; IV) product of strand transfer; V) DNA repair by cellular enzymes. Tn5 transposon: 1) donor DNA; 2) 3&apos;processing; 3–4) 5&apos; processing, consisting of loop formation (3) and generation of blunt-ended DNA (4); 5) strand transfer; 6) repaired strand transfer product. Portions of the donor DNA that become integrated are shown in red. Acceptor DNA is shown in white. Portions of acceptor DNA repaired following the strand transfer reaction are shown in grey.
- hepatitis C virus
- HIV integrase and HCV RNA dependent RNA polymerase (NS5B) share mechanistic similarities,requiring the presence of two divalent metal ions for their phosphoryltransferase activities. Both are inhibited by a,g-diketoacids (1) and (2). To avoid some of the issues associated with the diketoacids, we designed pyrimidine carboxylic acids (3) as replacements that share the same mode of inhibition of NS5B. However, these compounds still had low cell-based potency. The most likely cause of this low potency in cell systems is the highly polar nature of the pyrimidine carboxylic acids, which contain both a carboxylic acid and an acidic hydroxyl group at the 5-position [22]. Thus, replacements of both of these acidic functionalities were sought. Although the discovery of the dihydroxypyrimidines was in the context of a programme aimed at inhibiting HCV replication, given the shared mechanism, compounds were also tested on HIV integrase. It was not possible to replace the phenolic 5-hydroxyl of the dihydroxypyrimidine and retain inhibitory activity against the relevant enzymes. In the case of HCV NS5B, replacement of the carboxylic acid also proved extremely difficult. However, one of the potential acid replacements, a benzylamide (4), proved to be a potent inhibitor (IC50 85nM) of the strand transfer reaction catalysed by HIV integrase. This compound is inactive against HCV NS5B.
- This caused great excitement both at IRBM and in West Point. At the time the West Point group had worked on diketoacid integrase inhibitors and, understanding the limitations, had developed these into naphthyridine ketones (e.g., (5) Figure 1.2) [23]. Despite tremendous improvements in drug-like properties in comparison with the diketoacids, potency in this series in the cell-based assay remained an issue. The similarity between the pharmacophore of these naphthyridines and the pyrimidines was recognised, and led ultimately to the discovery of L-870812 and L-870810 in West Point. At IRBM, we chose to concentrate on lead compound (4). At the outset of the project we faced a number of issues: (4) had low activity in the cellbased assay, bioavailability in rat was low (15%), and the series was highly plasma protein bound. Plasma protein binding is a major issue for many HIV drugs (amongst others). The more a compound is bound to proteins in the blood, the less it is available for targeting the protein of interest, and hence the lower is the potency. This was addressed in the course of the programme by running the cell-based infectivity assay (Spread) [24] in the presence of low serum (10% foetal bovine serum, FBS), and also in the highest serum conditions feasible (50% normal human serum, NHS), and using the latter as a surrogate for whole blood activity. serum was useful as it highlighted whether or not compounds were intrinsically active in cells. Thus we could differentiate inherent low potency from that derived from high serum binding. The measurement used for the cell-based activity was IC95; the concentration of compound required to inhibit cell-based infectivity by at least 95% in the four-day multiple-cycle assay. The enzymatic assay we used to measure intrinsic in vitro activity was called Quickin (QI) [25]. This assay measured the ability of compounds to inhibit the strand transfer of a pre-processed DNA fragment into DNA (the second step catalysed by integrase). It has been shown that this class of molecules specifically inhibits this step. In this system, potency was quantified by measurement of IC50.
- DIHYDROXYPYRIMIDINES The most straightforward place to start lead optimisation of (4) was by modification of the benzylamide [26]. It was established that the NH was needed for activity (Table 1.1) (6), that at least one methylene was required (7), that the ring needed to be aromatic (8) and that phenethyl was marginally better than benzyl (9). Various heteroaromatic replacements for the phenyl ring (10, 11) were not improvements over phenyl, nor was a-branching (12). Generally, it was found that for methylene-linked heteroaryl substituents the potency correlated quite well with calculated log P. The search for more specific interactions involved extensive modification of the benzyl ring (Table 1.2).
- A para or meta substituent was preferred over ortho, and the most potent compounds came from substitution with a small electron-withdrawing group (e.g., fluorine) (22–24). In this case, the para substitution is preferred. Making the 4-substituent a larger halogen (25, 26) or a trifluoromethyl group (21) reduced activity. Other large (27), polar (28), acidic (29) or basic (30) groups were not tolerated. From this structure–activity relationship (SAR) it emerged that a 4-fluorobenzyl group was optimal for enzyme activity in this series. Later in the project this relationship was occasionally revisited, both to check that it was still true when various other changes were made and to potentially address other issues. For the most part this substituent was used throughout the course of the ongoing project.
- Various other changes were made to the central pyrimidine core of the molecule, and for the sake of brevity these are described as general conclusions in Figure 1.3. It is necessary to have a heteroatom at the 4-position of the pyrimidine, and an acidic hydroxyl at the 5-position. This is consistent with the model in which these atoms, along with the amide carbonyl, are necessary for binding to the metals in the catalytic centre of integrase. The nitrogen atoms are not vital, and as will be seen later, the nitrogen at the 1-position can be alkylated. The position with the most flexibility to make alterations is the 2-position, which in the lead bears a thiophene, but which became the focus of much of the remaining SAR. Although there are exceptions, many changes can be made at this position that do not have a great impact on intrinsic potency, but can be used to modulate physicochemical properties. In that way the cell penetration, protein binding, PK and other parameters of the compounds can be changed [27].
- This is well illustrated in Table 1.3, which shows the effect of a variety of 2-substituents on the enzyme activity. Removing the substituent completely (31) has only a small effect on enzyme activity, as does changing from an aromatic to alkyl (32, 33), changing the aromatic to a benzene ring (34), or moving the aromatic one carbon further from the pyrimidine ring (35).
- The prototypical example was compound (36) (Figure 1.4), in which a dimethylamino group was added to the benzylic methylene of (35). However, we also found that these compounds had low activity in our cell-based assays. For example (35) has activity weaker than 5 mM. To address this, one of the things that was tried was to introduce polar groups into the molecules. One type of polar group that we included was an amine, with the thinking that this could also help to ‘balance’ the charge on the molecules, as the 5-hydroxyl group is acidic. It was this change that proved the most beneficial and set us on a productive track, albeit that perhaps the rationale was not correct (our development compounds did not contain a basic amine). Although we saw some reduction in enzyme activity with (36), for the first time we saw a compound with similar activities in the enzymatic assay and the cell-based assay in the presence of low serum. This was an indication that (36) enters the cell effectively. A remaining issue, in terms of potency, was the low activity in the presence of the physiologically more relevant 50% normal human serum with a shift between low and high serum that correlated well with a measured human protein binding of W99%. Compound (36) had a very good PK profile with high oral bioavailability (rat, F 59%; dog, F 93%) and low clearance (rat 14 and dog 0.5 ml/min/kg). Given this profile, (36) became the lead on which we focused much of our effort, and in particular on improving cell-based activity in high serum conditions by reducing protein binding.
- It is well established [28] that, for lipophilic acids, binding to serum albumin correlates well with lipophilicity. That is also the case in this series and the correlation between measured logD and human protein binding is shown in Figure 1.5. This covers all of the compounds in the dihydroxypyrimidine class and the N-methylpyrimidones for which we have measured data.
- What is perhaps less well-precedented is the relationship between protein binding and the shift in a cell-based assay between low and high serum conditions. For compounds on this project, that relationship is shown in Figure 1.6. We found in the case of very high protein binding that, as expected, the shift was high. However, if protein binding was reduced to around 90% it was possible to obtain compounds with a low shift between the two serum conditions. Obviously at low protein binding the shift becomes small, but this is not optimal for the reason shown in Figure 1.7 which shows the relationship between measured rat protein binding and clearance in rats. Since metabolism plays a major role in clearance, and metabolism is structure dependent, one would not expect a good correlation of these simple parameters. Nonetheless, as a trend, it is clear that lower clearance is found for higher protein bound compounds. We cannot test the same correlation with human clearance, but the assumption is that it would be similar. From these analyses, it is clear that to improve cell-based activity in high serum in these compounds, one wants to reduce protein binding. To achieve this, the logD needs to be reduced. However, a careful balance needs to be struck, as it is free drug that is cleared in vivo. Thus very low protein binding tends to lead to high clearance. In our series of compounds the optimum comes in a protein binding range of around 80–90%, where it is possible to have compounds with both low shift and low clearance. Much of this analysis is post facto, but we were aware of the relationship between logD and protein binding from the outset, and sought to make (36) less protein bound by making it less lipophilic. Since we knew the aromatic ring of the 2-substituent was not important for enzyme activity, we removed it to give (37) (Table 1.4). Compound (37) maintained enzymatic activity, but potency was reduced three-fold in cells in low serum, indicating some loss of cell penetration. Reasoning that perhaps we had reduced the lipophilicity too far, we introduced a methyl group on the methylene (38) and saw approximately 10-fold improvement in the cell-based assay in low serum and, for the first time, achieved respectable sub-micromolar activity in the high serum conditions. Continuing on this theme, we added another methyl group, which had the added advantage of eliminating the chiral centre, to give (39). To our great excitement this proved very potent under both low and high serum conditions, with activity in the range that one would expect to be reasonable in a drug. Making the compound more lipophilic still (40) or having a free NH (41) were both detrimental.
- Compound (37) maintained enzymatic activity, but potency was reduced three-fold in cells in low serum, indicating some loss of cell penetration. Reasoning that perhaps we had reduced the lipophilicity too far, we introduced a methyl group on the methylene (38) and saw approximately 10-fold improvement in the cell-based assay in low serum and, for the first time, achieved respectable sub-micromolar activity in the high serum conditions. Continuing on this theme, we added another methyl group, which had the added advantage of eliminating the chiral centre, to give (39). To our great excitement this proved very potent under both low and high serum conditions, with activity in the range that one would expect to be reasonable in a drug. Making the compound more lipophilic still (40) or having a free NH (41) were both detrimental.
- The PK properties of (39) in three preclinical species were very good (Table 1.5) with good to excellent oral bioavailability, low to moderate clearance and reasonable half lives. Given that we were now making compounds with potency and PK that could be suitable for development, we were interested in predicting human PK. To do this, we studied the routes and rates of metabolism in animals and in in vitro systems, including human. Also, given the importance of co-dosing of HIV drugs, it was important to avoid drug–drug interactions as far as possible. At the very least we did not want our compound to be a perpetrator of these interactions, and so a lack of inhibition of the major cytochrome P450s was an important objective. Compound (39) is not an inhibitor of CYP450s, nor an inducer of CYP3A4. It was not metabolised oxidatively (no turnover in liver microsomes in the presence of NAPDH), and the major metabolite seen in animals, and in liver microsomes in the presence of uridine diphosphate glucuronic acid (UDPGA), was the 5-O-glucuronide. Comparing the rates of glucuronidation in animals and man (Figure 1.8), and taking into account protein binding (rat 83%, dog 91% and human 89%) we predicted that human PK should be similar to that seen in dog.
- In addition to simple acyclic substituents at the 2-position of the pyrimidine ring, we explored constraining the basic amino group into a ring (Table 1.6). The best position for the basic nitrogen was at the 2-position of the saturated heterocycle [(42) compared with (43) and (44)] although cell-based activity with a free NH was not good for any of these compounds. Methylation of the basic nitrogen gave a compound (45) with much improved activity in the cell-based assay, and the 5-membered analogue (46) was similarly potent.
- At this point an important observation was made. We had investigated the effect of methylation of the pyrimidine 2-nitrogen for a small number of compounds, including (50), the analogue of (45). Key attributes of these two compounds are shown in Table 1.7. Although the N-Me pyrimidinone (50) had lower potency, both on the enzyme and in the cell-based assays, than the dihydroxypyrimidine (45), it had two important improvements. The first was that the protein binding was considerably lower (a factor that we had struggled with in the dihydroxypyrimidine series) and the second was that rat PK was improved, with better oral bioavailability and lower plasma clearance. We reasoned that it would be easier to improve potency in a series with good protein binding and PK than the converse and turned our attention to the N-Me pyrimidinone series.
- As well as a 2-substituted piperidine at the 2-position of the pyrimidine, we also explored the 5-membered ring analogues. The N-methylpyrrolidine (47) (Table 1.8) was slightly more potent on the enzyme than (50), but was less active in the cell-based assay. Various substitutions around the pyrrolidine (data not shown) showed that the 4-position was optimal for substitution, in particular with small electron-withdrawing substituents. For example, the methoxy (48) and fluoro (49) substituted compounds had cell-based potencies close to the range in which we were ultimately interested. An option available in the piperidine series that was not realistic for the pyrrolidines, was to incorporate a heteroatom into the ring, and results for morpholine (51), thiomorpholine (52) and piperazine (53) are shown. As for the exocyclic substituents in the pyrrolidines, these gave a nice increase in cell-based activity, with the morpholine (51) being the most potent in high serum conditions. Resolution gave the separate enantiomers [(+)-(54) and ()-(55)]. The former proved slightly more active than the latter, and was fully profiled in PK and other preclinical experiments. The PK profile of (54) is shown in Table 1.9. The compound showed good oral bioavailability, low to moderate clearance and reasonable half lives. It was not an inhibitor of the major human CYP450s (3A4, 2D6, 1A2 and 2C19), and was not metabolised oxidatively but by glucuronidation. Based on the rate of turnover in liver microsomes in the presence of
- Lost permeability?? UDPGA, we again predicted that human clearance would be similar to that of dog. Based on the fact that we could find very interesting compounds in this N-methylpyrimidine series, we went on to explore simpler, acyclic substituents at the 2-position. Our first forays into this area were not successful. Taking the best substituent from the dihydroxypyrimidines and applying it to the N-methylpyrimidones gave a compound (56) (Table 1.10) which had less than the desired activity in both enzyme and cell-based assays. Taking away one methyl group (57), both methyl groups from the basic nitrogen (58), or all methyl groups on this substituent (59), gave no improvement in cell-based activity.
- We were well aware that neither L-870812 nor L-870810 had basic nitrogen atoms and thus it seemed that it would be possible to make compounds in this series that did not have this basic nitrogen. Compound (58) was therefore acylated, sulphonylated and so on, to give compounds with polar but not basic substituents at the 2-position (Table 1.11). With a simple acyl group on the nitrogen (60), we started to recover cellbased activity, albeit not to the desired level. Trifluoromethyl (61) was not an improvement, and although a sulphonamide (62) or sulphamide (63) gave good activity in low serum conditions, the serum shift was moderate to high.
- Neither urea (64) nor oxalamide (65) had good cell-based activity, but replacing the ester in (65) with a dimethylamide (66) gave a very interesting compound. The PK properties of (66) are shown in Table 1.12. The compound showed moderate to good oral bioavailability, low to moderate clearance and reasonable terminal half lives (elimination was multiphasic in all species, and the half life of the terminal phase is shown). It was not an inhibitor of the major human CYP450s, nor of uridine diphosphate glucuronyl transferase 1A1(UGT1A1), and was not metabolised oxidatively but by glucuronidation, predominantly by UGT1A1. Based on the in vivo data, the rate of turnover in liver microsomes in the presence of UDPGA (rat, dog, rhesus and human 20, 3, 46 and 8 ml/min/mg, respectively) and the free fraction in animal and human plasma (rat, dog, rhesus and human 35, 38, 18 and 28%, respectively), we predicted human clearance would be low to moderate.
- piperidine
- Bearing in mind the importance of avoiding, as far as possible, clinical resistance to this new class of agents, during the process of fully characterising (66), we profiled it against a panel of resistant mutants. These were mutant viruses which had been raised during the course of many years research using a number of compounds from various classes to generate the resistance mutations. The results in these assays for (66) and for various other compounds are shown in Table 1.13. As compared to L-870810, (66) showed a somewhat less good profile across these resistant mutants. It was our thinking that although this is not proven, it was likely that a compound with more shift versus wild type in its resistance profile was likely to be associated with a lower genetic barrier to generating resistant mutants in vivo. Whilst the profile of (66) was not hugely worse than L-870810, and considerably better than S-1360, given the importance of resistance mutations clinically, we wanted to do everything possible to avoid generating resistance in patients. It was particularly concerning that a single point mutant (F121Y) which was not replicationimpaired gave a more than 10-fold shift with (66). We therefore sought compounds as good as or better than (66) in preclinical profile, with an improved profile on the integrase mutants. At this point in the project we had a number of compounds which had potencies in the range of 100nM or better in the cell-based assay in high serum, and reasonable to good rat PKs. The approach we took was to screen all such compounds against the panel of mutants, looking for those that had improved resistance profiles. It is beyond the scope of this chapter to go into all those compounds, but I touch on a particular class which proved very fruitful.
- As part of ongoing SAR studies we looked to replace the dimethylamide of (66) with isosteres that may give advantage. In particular, we were interested in small aromatic heterocycles (Table 1.14) as these may maintain the electronic properties of the carboxamide while changing physicochemical properties or some other feature of the molecule. A six membered heterocycle (67) with one heteroatom had lower activity than desired in the cell-based assay, but introducing a second heteroatom (68) gave very interesting levels of potency.
- In the five-membered heterocycles, three heteroatoms (70) were better than two (69), and (70) had outstanding potency in the cell-based assay. As is common throughout this work, making the compound too polar (71) led to a loss of cell-based potency under low serum conditions presumably due to low cell permeability, and making the compound too lipophilic (72) led to larger shifts under high serum conditions due to high protein binding. From all of this work, (70) emerged as the best compound. Compound (70) came to be known as MK-0518 and latterly as raltegravir. On the basis of its potency, and the resistance profile shown in Table 1.13, (70) was fully profiled in preclinical screens. It had good oral bioavailability in rats and dogs (Table 1.15). In monkeys bioavailability was rather lower, but this may have been due to the salt form used – in rats and dogs the potassium salt was dosed, whereas we used the free acid in monkeys. Clearance was low to moderate, and again the compound showed multiphasic elimination in all preclinical species, with terminal half lives that were long. It was not an inhibitor of the major CYP450s, did not induce CYP3A4 in human hepatocytes and it was not an inhibitor of UGT1A1. Thus it has a low probability to be a perpetrator of drug–drug interactions. In rats, clearance was mainly metabolic, with little parent seen in urine and bile. The clearance was largely via glucuronidation with little oxidative metabolism seen in vitro and in vivo. The enzyme mainly responsible for glucuronidation was UGT1A1, although some turnover was also seen with other UGTs. Based on the in vivo data, the rates of turnover in microsomes in the presence of UDPGA (rat, dog, rhesus and human 34, 2, 36 and 9 ml/min/mg, respectively) and the free fraction in animal and human plasma (rat, dog, rhesus and human 27, 29, 15 and 17%, respectively), we predicted that human clearance would be low to moderate. It is not the place of this review to summarise the further safety assessment and clinical findings with raltegravir, but suffice it to say that the compound has been generally well tolerated in man, and has shown excellent efficacy both in short-term monotherapy studies [29] and also in longer term studies in combination with other antiretrovirals [30].
- In the same way, integrase inhibitors bind to integrase, the enzyme HIV uses to insert its newly made genetic material (DNA) into a CD4 cell&apos;s DNA. Their development marks an important frontier in HIV research, since integrase is one of the only enzymes that hasn&apos;t yet been successfully targeted. After ten years of attempts to design an integrase inhibitor with little success (including L-870810, zintevir, and S-1360), research presented at the 13th Conference on Retroviruses and Opportunistic Infections (CROI) finally shows two candidates, MK-518 and GS-9317, that could be the first successful integrase inhibitors.
- Potassium salts of compd. I, which are HIV integrase inhibitors useful for treating or prophylaxis of HIV infection, for delaying the onset of AIDS, and for treating or prophylaxis of AIDS, were prepd. Thus, successive Strecker amine formation of acetone cyanohydrin with ammonia (97%), N-protection with CbzCl (86%), nucleophilic addn. with hydroxylamine (88%), cyclocondensation with di-Me acetylenedicarboxylate (51.7%), N-methylation of the resultant dihydropyrimidinecarboxylic Me ester with MeI (70%), amidation of the ester with p-fluorobenzylamine (90%), deprotection with H2 in the presence of Pd/C (96%), and acylation with 5-methyl-1,3,4-oxadiazole-2-carbonyl chloride (prepn. given) (91%) afforded I. Conversion of I into its monopotassium salts was achieved with KOH or KOEt in different solvents, and the salts were characterized by X-ray powder diffraction patterns and differential scanning calorimetry (DSC) curves. From an understanding of mechanism of related enzymes (HCV polymerase and HIV integrase) in distinct viruses, came the insight and decision to test compounds made as inhibitors of one on the other. This led to both a breakthrough in terms of potency in one series and to a novel series of inhibitors of HIV integrase. Although active in the enzymatic assay, this novel series was insufficiently active in cell-based assays. Altering the physicochemical properties allowed the compounds to enter cells. However, early compounds were not sufficiently active under the physiologically relevant conditions due to high protein binding. This was addressed using an understanding of how physicochemical properties affect protein binding. It was then a balancing act, keeping protein binding low enough to ensure good cell-based activity, whilst not so low as to lead to high plasma clearance. An important decision point in the project was to switch interest from dihydroxypyrimidines to N-methylpyrimidones: although earlier examples of the latter were not as potent, in our minds the advantages of lower protein binding and better PK outweighed this drawback, and the series was optimised back to highly potent compounds with good PK properties. Keeping an eye open to potential issues in clinical use, attention was paid throughout the project to minimising the risk of drug–drug interactions by making sure we did not make compounds that were not metabolised by, and did not inhibit or induce cytochrome P450s. In a similar vein, we recognised the importance of resistance to clinically used antiretrovirals, and strove to maintain the best profile we could against those mutant enzymes available at the time that were resistant to related compounds with this mechanism of inhibition. The end result of this project was raltegravir, which has shown safety and efficacy profiles in clinical studies that are very promising indeed, and which should offer a new treatment option and renewed hope to sufferers of this important disease.
- In addition to L-870810 from Merck, a number of other companies have had programmes on integrase inhibitors and clinical data is available for some of them. An early entrant in the field was S-1360 (Figure 1.9) [31] which GlaxoSmithKline licensed from Shionogi. It is a diketoacid derivative, in which the acid has been replaced with a triazole. It has moderate activity in a cell-based assay (EC90) around 1 mM. In a Phase 1 study the compound was generally well tolerated with good PK, but after an efficacy study in Phase II the compound was discontinued. Gilead have reported [32] Phase II data on elvitegravir (GS-9137, JT-303) licensed from Japan Tobacco. It is metabolised in part by CYP3A4, and thus exposure after oral dosing is increased by co-dosing with ritonavir (an HIV protease inhibitor that is also an inhibitor of CYP3A4), and it is this combination that has been in Phase II trials as a once-daily dose. In the Phase II studies, elvitegravir was generally well tolerated. A dose of 125 mg elvitegravir/100 mg ritonavir in combination with two nucleosides was superior to a boosted HIV protease inhibitor-containing regimen. GlaxoSmithKline have reported [33] Phase I data for GSK364735. The compound has protein-adjusted EC90 of 0.062 mg/ml, and various dosing regimes gave plasma levels which exceeded that target. It was generally well tolerated and did not have CYP450 mediated interactions. The structure is not publicly available. Various other companies have been active in the area of integrase inhibitors, including Bristol Myers Squibb with patents on compounds similar to the pyrimidinone structure of raltegravir.
- Detailed Description: While highly active antiretroviral therapy (HAART) reduces plasma HIV-1 levels to below the limits of detection with standard assays, replication-competent virus persist in a stable, latent reservoir in resting CD4+ T cells. So, there is a rapid resumption in plasma viremia when therapy is interrupted. In addition to cellular reservoir, other pharmacologically privileged areas such as the central nervous system and the genital tract might act as additional sources of residual virus in patients with undetectable levels of plasma HIV-1 RNA. There is great current interest in strategies for depleting and eliminating this reservoir. The antiviral potency of current regimens emerges as an important determinant of complete viral control. In certain patients, the latent reservoir decay can be hastened with treatment intensification. An intensification with the HIV-1 integrase inhibitor Raltegravir (RAL) of a stable HAART regimen with persistent HIV-1 viral suppression could increase the slope of decay of the HIV-1 latent reservoir. This could provide further insight into this area, decrease the size of latent reservoir, and translate into clinical benefits for patients being simplified to maintenance monotherapy with RAL or in the HIV-1 rebound kinetics and slope after a programmed treatment interruption. When HIV has just been made, it enters a CD4 cell&apos;s nucleus. An enzyme in the nucleus called integrase helps HIV hide in the cell&apos;s own DNA, and another enzyme called histone deacetylase (HDAC) helps it stay hidden. While HIV is hiding in the DNA, it is not active and cannot be targeted by currently available highly active antiretroviral therapy (HAART). VPA, a drug used to treat seizures, is an HDAC inhibitor, and raltegravir is a newly FDA-approved integrase inhibitor. Taking VPA and raltegravir may prevent HIV from being able to hide in CD4 cells, allowing HAART to eliminate HIV that would normally be hidden. The purpose of this study is to determine whether adding raltegravir and VPAto the participant&apos;s current HAART regimen will reduce the number of resting HIV in CD4 cells.