pomalidomide is 2 mg daily compared to 25 mg for lenalidomide, leading to 10-100 times lower plasma drug concentration of pomalidomide. t-cell activation thalidomide and its analogs help with the co-stimulation of t-cells through the b7-cd28 complex by phosphorylating tyrosine on the cd28 receptor. in vitro data suggests this co-stimulation leads to increased th1 type cytokine release of ifn-γ and il-2 that further stimulates clonal t cell proliferation and natural killer cell proliferation and activity. this enhances natural and antibody dependent cellular cytotoxicity. lenalidomide and pomalidomide are about 100-1000 times more potent in stimulating t-cell clonal proliferation than thalidomide. in addition, in vitro data suggests pomalidomide reverts th2 cells into th1 by enhancing transcription factor t-bet. anti-angiogenesis angiogenesis or the growth of new blood vessels has been reported to correspond with mm progression where vascular endothelial growth factor (vegf) and its receptor, bfgf and il-6 appear to be required for endothelial cell migration during angiogenesis. thalidomide and its analogs are believed to suppress angiogenesis through modulation of the above-mentioned factors where potency in anti-angiogenic activity for lenalidomide and pomalidomide was 2-3 times higher than for thalidomide in various in vivo assays, thalidomide has also been shown to block nf-κb activity through the blocking of il-6, and nf-κb has been shown to be involved in angiogenesis. inhibition of tnf-α is not the mechanism of thalidomide's inhibition of angiogenesis since numerous other tnf-α inhibitors do not inhibit angiogenesis. anti-tumor activity in vivo anti-tumor activity of thalidomide is believed to be due to the potent anti-angiogenic effect and also through changes in cytokine expression. in vitro assays on apoptosis in mm cells have been shown, when treated with thalidomide and its analogs, to upregulate the activity of caspase-8. this causes cross talking of apoptotic signaling between caspase-8 and caspase-9 leading to indirect upregulation of caspase-9 activity. further anti-tumor activity is mediated through the inhibition of apoptosis protein-2 and pro-survival effects of igf-1, increasing sensitivity to fas mediated cell death and enhancement of tnf-related apoptosis inducing ligand. they have also been shown to cause dose dependent g0/g1 cell cycle arrest in leukemia cell lines where the analogs showed 100 times more potency than thalidomide. bone marrow environment the role of angiogenesis in the support of myeloma was first discovered by vacca in 1994. they discovered increased bone marrow angiogenesis correlates with myeloma growth and supporting stromal cells are a significant source for angiogenic molecules in myeloma. this is believed to be a main component of the mechanism in vivo by which thalidomide inhibits multiple myeloma. additionally, inflammatory responses within the bone marrow are believed to foster many hematological diseases. the secretion of il-6 by bone marrow stromal cells (bmsc) and the secretion of the adhesion molecules vcam-1, icam-1 and lfa, is induced in the presence of tnf-α and the adhesion of mm cells to bmsc. in vitro proliferation of mm cell lines and inhibition of fas-mediated apoptosis is promoted by il-6. thalidomide and its analogs directly decrease the up-regulation of il-6 and indirectly through tnf-α, thereby reducing the secretion of adhesion molecules leading to fewer mm cells adhering to bmsc. osteoclasts become highly active during mm, leading to bone resorption and secretion of various mm survival factors. they decrease the levels of adhesion molecules paramount to osteoclast activation, decrease the formation of the cells that form osteoclasts and downregulate cathepsin k, an important cysteine protease expressed in osteoclasts. structure-activity relationship figure 5: thalidomide with the ring system outlined since the mechanism of action of thalidomide and its analogs is not fully clear and the bioreceptor for these substances has not been identified, the insight into the relationship between the structure and activity of thalidomide and its analogs are mostly derived from molecular modelling and continued research investigation. the information on sar of thalidomide and its analogs is still in process so any trends detailed here are observed during individual studies. research has mainly focused on improving the tnf-α and pde4 inhibition of thalidomide, as well as the anti-angiogenesis activity. tnf-α inhibitors (not via pde4) research indicated that a substitution at the phthaloyl ring would increase tnf-α inhibition activity (figure 5). an amino group substitution was tested at various locations on the phthaloyl ring (c4, c5, c6, c7) of thalidomide and em-12 (previously described). amino addition at the c4 location on both thalidomide and em-12 resulted in much more potent inhibition of tnf-α. this also revealed that the amino group needed to be directly opposite the carbonyl group on the isoindolinone ring system for the most potent activity. these analogs do not inhibit pde4 and therefore do not act by pde4 inhibition. other additions of longer and bigger groups at the c4 and c5 position of the phthaloyl ring system of thalidomide, some with an olefin functionality, have been tested with various results. increased inhibitory effect, compared to thalidomide, was noticed with the groups that had an oxygen atom attached directly to the c5 or c4 olefin. iodine and bromine addition at c4 or c5 resulted in equal or decreased activity compared to thalidomide. these groups were not compared with lenalidomide or pomalidomide. pde4 inhibitors figure 6: rolipram, highlighting the 3,4-dialkoxyphenyl moiety figure 7: common structure for pde4-inhibiting thalidomide analogs the common structure for analogs that inhibit tnf-α via inhibition of pde4 is prepared on the basis of hydrolysing the glutarimide ring of thalidomide. these analogs do not have an acidic chiral hydrogen, unlike thalidomide, and would therefore be expected to be chirally stable. on the phenyl ring, a 3,4-dialkoxyphenyl moiety (figure 6) is a known pharmacophore in pde4 inhibitors such as rolipram. optimal activity is achieved with a methoxy group at the 4-position (x2) and a bigger group, such as cyclopentoxy at the 3-position carbon (x3). however the thalidomide pde4 inhibitory analogs do not follow the sar of rolipram analogs directly. for thalidomide analogs, an ethoxy group at x3 and a methoxy group at x2, with x1 being just a hydrogen, gave the highest pde4 and tnf-α inhibition. substitutes larger than diethoxy at the x2–x3 position had decreased activity. the effects of these substitutions seem to be mediated by steric effects. for the y-position, a number of groups have been explored. substituted amides that were larger than methylamide (conhch3) decrease pde4 inhibition activity. using a carboxylic acid as a starting point, an amide group has similar pde4 inhibition activity but both groups were shown to be a considerably less potent than a methyl ester group, which had about six-fold increase in pde4 inhibitory activity. sulfone group had similar pde4 inhibition as the methyl ester group. the best pde4 inhibition was observed when a nitrile group was attached, which has 32 times more pde4 inhibitory activity than the carboxyl acid. substituents at y leading to increasing pde4 inhibitory activity thus followed the order: cooh ≤ conh2 ≤ cooch3 ≤ so2ch3 < cn substitutions on the phthaloyl ring have been explored and it was noticed that nitro groups at the c4 or c5 location decreased activity but c4 or c5 amino substitution increased it dramatically. when the substitution at the 4 (z) location on the phthaloyl ring was examined, hydroxyl and methoxy groups seem to make the analog a less potent pde4 inhibitor. an increase in activity was observed with amino and dimethylamino to a similar extent but a methyl group improved the activity further than the aforementioned groups. a 4-n-acetylamino group had slightly lower pde4 inhibitory activity, compared with the methyl group, but increased the compound's tnf-α inhibitory activity to a further extent. substituents at z leading to increasing pde4 inhibitory activity thus followed the order: n(ch3)2 ≤ nh2 < nhc(o)ch3 < ch3 angiogenesis inhibition figure 8: common structure for thalidomide analogs with angiogenesis inhibition for angiogenesis inhibition activity, an intact glutarimide ring seems to be required. different groups were tested in the r position. the substances that had nitrogen salts as the r group showed good activity. the improved angiogenesis inhibitory activity could be due to increased solubility or that the positively charged nitrogen has added interaction with the active site. tetrafluorination of the phthaloyl ring seems to increase the angiogenesis inhibition. synthesis described below are schemes for synthesizing thalidomide, lenalidomide, and pomalidomide, as reported from prominent primary literature. note that these synthesis schemes do not necessarily reflect the organic synthesis strategies used to synthesize these single chemical entities. thalidomide scheme 1: thalidomide synthesis, the older procedure scheme 2: newer thalidomide synthesis, two step reaction synthesis of thalidomide has usually been performed as seen in scheme 1. this synthesis is a reasonably simplistic three step process. the downside of this process however is that the last step requires a high-temperature melt reaction which demands multiple recrystallizations and is not compliant with standard equipment. scheme 2 is the newer synthesis route which was designed to make the reaction more direct and to produce better yields. this route uses l-glutamine rather than l-glutamic acid as a starting material and by letting it react with n-carbethoxyphthalimide gives n-phthaloyl-l-glutamine (4), with 50–70% yield. the substance 4 is then stirred in a mixture with carbonyldiimidazole (cdi) with enough 4-dimethylaminopyridine (dmap) in tetrahydrofuran (thf) to catalyze the reaction and heated to reflux for 15–18 hours. during the reflux thalidomide crystallizes out of the mixture. the final step gives 85–93% yield of thalidomide, bringing the total yield to 43–63%. lenalidomide and pomalidomide scheme 3: pomalidomide synthesis both of the amino analogs are prepared from the condensation of 3-aminopiperidine-2,6-dione hydrochloride (compound 3) which is synthesized in a two step reaction from commercially available cbz-l-glutamine. the cbz-l-glutamine is treated with cdi in refluxing thf to yield cbz-aminoglutarimide. to remove the cbz protecting group hydrogenolysis, under 50–60 psi of hydrogen with 10% pd/c mixed with ethyl acetate and hcl, was performed. the formulated hydrochloride (compound 3 in scheme 3) was then reacted with 3-nitrophthalic anhydride in refluxing acetic acid to yield the 4-nitro substituted thalidomide analog and the nitro group then reduced with hydrogenation to give pomalidomide. scheme 4: lenalidomide synthesis lenalidomide is synthesized in a similar way using compound 3 (3-aminopiperidine-2,6-dione) treated with a nitro-substituted methyl 2-(bromomethyl) benzoate, and hydrogenation of the nitro group. pharmacokinetics thalidomide main article: thalidomide thalidomide tmax 4–6 hours in subjects with mm protein binding 55–65% metabolites hydrolized metabolites half-life 5.5–7.6 hours lenalidomide main article: lenalidomide lenalidomide tmax 0.6–1.5 hours in healthy subjects 0.5–4 hours in subjects with mm protein binding ~30% metabolites has not yet been studied half-life 3 hours in healthy subjects3.1–4.2 hours in subjects with mm pomalidomide main article: pomalidomide pomalidomide tmax 0.5–8 hours protein binding unknown metabolites unknown half-life 6.2–7.9 hours