Thienopyridinones And Thienopyrimidinones

The first potent and orally active human GnRH receptor antagonist, T-98475 (1), was reported in 1998 [9]. It inhibits [125I]leuprorelin binding to the cloned human GnRH receptor with an IC50 value of 0.2 nM. It also has potent binding affinity to membrane fractions of monkey pituitary with an IC50 of 4 nM, but somewhat lower activity on the rat pituitary (IC50 = 60 nM). It suppresses plasma LH concentration up to 25% level after oral administration in castrated male cynomolgus monkeys (60 mg/kg). A small set of SAR data suggested that the benzyl side-chain on the basic nitrogen contributes a 30-fold increase in binding affinity.

A slightly different series of compounds from T-98475, which removes the metabolically labile ester group, has been reported recently [10]. A representative compound from this series TAK-013 (2) binds with high affinity to human or monkey receptors expressed in CHO cells (IC50 values of 0.1 and 0.6 nM, respectively). Functionally, TAK-013 inhibits GnRH-stimulated arachidonic acid release with IC50

values of 0.06 and 10 nM, for human and monkey receptors, respectively. In cynomolgus monkeys, after oral doses of 10 mg/kg, TAK-013 reaches maximal concentration of 140 ng/ml at 6 h with an AUC of 568 ng h/ml. This good oral exposure enables TAK-013 to almost completely suppress plasma LH levels (11% of pretreatment at 24 h) after oral administration of a 30 mg/kg dose in castrated male cynomolgus monkeys. Chronic administration (30 mg/kg, three times daily) over the course of four menstrual cycles in normal cycling female cynomolgus macaques resulted in a suppression of LH, estradiol and progesterone, but not FSH [11]. TAK-013 is highly lipophilic and poorly soluble in water with a log D value higher than 4. Structure-activity studies on the side-chain of the basic nitrogen reveals the benzyl group can be replaced by a more polar and slightly basic 2-pyridylmethyl group resulting in a 20-fold improvement in binding affinity (Ki values are 180 and 9 nM for 3 and 4, respectively) [12].

5 6 7


An initial lead compound 5 was obtained from high throughput screening, and the IC50 of 5 at the rat GnRH receptor is reported to be 3 mM [13]. SAR studies of this series resulted in potent GnRH antagonists such as 6 with IC50 values of 60 nM on the rat GnRH receptor and 2.5 nM at the cloned human GnRH receptor [14-16]. Differences in affinities for GnRH receptors from different species have been reported for this and all other series of nonpeptide GnRH antagonists studied thus far. Functionally, 6 inhibits GnRH-stimulated LH release from rat pituitary cells with an IC50 of 85 nM, and GnRH-stimulated inositol phosphate hydrolysis in CHO cells expressing the human GnRH receptor (IC50 = 16 nM). A more potent human GnRH antagonist 7 (hGnRH IC50 = 0.44 nM, PI turnover IC50 = 1.0 nM) also binds very tightly to the rhesus monkey GnRH receptor (IC50 = 0.5 nM) and rat GnRH receptor (IC50 = 4.0 nM), but less at the dog GnRH receptor (IC50 = 60 nM). This compound suppresses circulating LH and testosterone levels in male rhesus macaques when given intravenously (0.5 mg/kg) [17].


Initial SAR studies on a lead compound with a 2-aryltryptamine structure (8, IC50 = 3 mM) discovered from high throughput screening suggests that the optimal distance between the 4-phenol group and the basic amine is a four-carbon linkage [18]. When the 3,4-dimethoxyphenyl at the 2-position of tryptamine core is replaced with a 3,5-disubstituted phenyl, the binding affinity increases. For example, the 3,5-dimethoxy, 3,5-dichloro, and 3,5-dimethylphenyl analogs have IC50 values of 700, 170 and 50 nM, respectively. The dimethylphenyl compound (9) is over 25-fold better than the unsubstituted phenyl analog (IC50 = 1.3 mM). However, the corresponding 4-fluorophenyl analog is about eightfold less active which suggests a lipophilic electron-rich phenyl group is favored at this position for interaction with the GnRH receptor, possibly at an aromatic residue. Further SAR study reveals a lipophilic group with hydrogen bonding capability at the 5-position of the tryptamine core improves binding affinity about 10-fold. Thus, 10 has an IC50 value of 4 nM [19]. When the OH group of the phenol was replaced with a methanesulfonamido group, the binding affinity further improved (11, IC50 = 7 nM). While the binding data above were obtained from crude membranes prepared from rat pituitary glands, the binding affinity of these compounds is less potent at the human GnRH receptor. For example, 11 exhibits an IC50 value of 170 nM at the cloned human receptor, an over 40-fold difference [20]. The phenol can also be replaced by a heteroaromatic ring with hydrogen bonding capability. Thus, the 4-pyridyl analog 12 has an IC50 value of 41 nM at the rat GnRH receptor [21].

17: R = A= N

Several very potent GnRH antagonists were synthesized by a combination of the previous SAR information. Thus, compounds with a 5-amide bulky group exhibit low nanomolar binding affinity at the rat receptor (IC50 values for 13, 14 and 15 are 6, 3 and 2 nM, respectively). Functionally, they inhibit LH release from rat pituitary glands with IC50 values of 540, 72 and 42 nM. These data suggest the sulphonamides 14 and 15 are much more functionally active than the phenol such as 13. Interestingly, the binding affinity of this series of compounds is greatly improved by the presence of 0.1% BSA in the binding assay, and the IC50 values obtained under this condition are 10-fold more potent. Thus, the IC50 values for 13,14 and 15 are now 0.5, 0.3 and 0.2 nM, respectively. The authors suggest that this shift is due to BSA preventing nonspecific loss of compound to glass assay tubes [22].

The function of the 5-amido group of the tryptamine seems not only to increase binding affinity of this series of compounds at the rat GnRH receptor, but also to narrow the gap in binding affinities between the rat and human GnRH receptors. For example, 16 has IC50 values of 0.6 and 7.1 nM at the rat and human GnRH receptors, respectively, which is only a 12-fold difference [23]. On the other hand, 11, without a 5-substitution, has a 40-fold difference between these two species. Compound 17 is a very potent human GnRH receptor antagonist with an IC50 of 1.4 nM in receptor binding, and IC50 of 18 nM in inhibition of GnRH-stimulated inositol phosphate hydrolysis.

Because of the enhancement of both binding and function of this series of compounds from the substitution at the 5-position together with the introduction of (S)-methyl group at 2-aminoethyl side chain of the tryptamine core, the linker between the basic nitrogen and the aromatic side-chain can be reduced to 2-carbon without loss of activity (18a, IC50 = 0.7 nM and 18b, IC50 = 0.8 nM). In contrast, the corresponding 2-carbon version of 12 is about 12-fold less active. This series of compounds have been further modified to improve pharmacokinetic profiles. In dogs, 19b (IC50 = 0.8 nM) has an oral bioavailability of 25% and an elimination half-life of 3.9 h. It has a similar profile in monkeys (F = 21%, t1=2 = 3.3 h), but is less bioavailable in rats (F = 8%, t1=2 = 1 h) [24]. Introduction of a methyl group at the 2-position of the pyridine to reduce both 2- and N-oxidations of pyridine, improved oral bioavailability in dogs (19c, F = 36%) but the 2-hydroxymethylpyridine analog 19d with excellent in vitro profile did not improve bioavailability in dogs (F = 15%) [25]. Despite limited oral bioavailability 19b (IC50 = 1.7 nM, rat receptor) dose-depen-dently inhibits LH release for periods ranging from 5 to 7 h at 5 mg/kg po and > 15 h at 20 mg/kg po in castrated male rats. Among compounds with different bicyclic side-chains, 19f exhibits excellent oral bioavailability in dogs (F = 63%), while the very close analog 19e has much lower oral exposure (F = 16%). Furthermore, low oral bioavailability of 19g (F = 5%) was observed [26]. The authors speculate that the urea structure of 19g has very strong hydrogen-bonding capability that reduces cell permeability. Unlike most compounds in this series, 19h has less risk of inhibition of cytochrome P450 3A4 (IC50 = 7.5 mM) while maintaining potent GnRH binding affinity (IC50 = 0.3 nM). This compound was also shown to be efficacious in the castrated male rats, where it reduced plasma LH levels for 14 h after a single oral dose at 10 mg/kg.

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