904 Current Organie Chemistry,2006.Vol.10.No. Poterat and Hamburge preparative HPLC. 14 toa main peak at 16.1 min (FHPLC-UV-MS nalysis all effec A typical example is shown in Fig 4 with vity of a lipophili (MAO)A NO med use cular dMAO A ayed surement (8)or resistomycin (9). of the poir ctio (315 an Among other hits,n family wed of MAO A inhib e of ds was econtain spiking crude hibiting strong UVVis siton isfor fuorescence. Thethyosine kinase activity of humane otent compo HPLC-Based profiling in Industrial environment HPLC-based d in A similar an of a in the HTS development and the omplete work-up of alla activ extract o the stems and of the shru en HPL which is usually tricted to as less as ofiling.Acti ity profile and ic data e stored in th nout need of mac aloid (1)with potent antagonist 904 Current Organic Chemistry, 2006, Vol. 10, No. 8 Potterat and Hamburger the overall effect. A typical example is shown in Fig. 4 with the tracking of the inhibitory activity of a lipophilic extract of Salvia miltiorrhiza Bunge (Lamiaceae) on recombinant monoamine oxidase (MAO) A and inducible NO synthase (iNOS) [49]. S. miltiorrhiza, known as “Danshen”, is a renowned Chinese medicinal plant chiefly used in the treatment of various cardiovascular disorders and in some infectious and inflammatory diseases. MAO A activity was assayed with a kinetic measurement of the conversion of kynuramine to 4-hydroxyquinoline [50]. Correlation of the activity profiles with the HPLC fingerprint strongly pointed towards the tanshinone-type diterpenoids as compounds with dual inhibitory properties for MAO A and iNOS induction. Targeted preparative purification afforded tanshinone IIA (3), 15,16-dihydrotanshinone I (4), cryptotanshinone (5) and tanshinone I (6). Dihydrotanshinone I was in both assays the most potent compound (MAO A: IC50 23 µM; iNOS: IC50 2.4 µM) while tanshinone IIA showed only marginal activity. The robustness of the kinetic assay for the detection of MAO A inhibition in the presence of potentially interfering compounds was established by spiking crude plant extracts, selected to contain diverse compounds exhibiting strong UV/Vis absorption and/or fluorescence, with small amounts of known inhibitors [50]. HPLC-Based Profiling in Industrial Environment HPLC-based activity profiling has been widely used in natural product HTS programs to prioritize hits and guide isolation work. Prioritization is of utmost importance in a HTS environment, since screens not seldom deliver a large number of hits exceeding by far the isolation capacities. A complete work-up of all active extracts is impossible within the timeframe allocated to follow-up activities in the lead discovery phase, which is usually restricted to as less as a few weeks. As a major asset, the microfractionation step can be performed with the bioactive sample stored in the library, without need of macroscopic isolation nor time-consuming refermentation or recollection activities and the associated issues of unsatisfactory reproducibility. With the activity profile at hand, isolation capacities can be dedicated to extracts where bioactivity correlates with chromatographic peaks. Preparative purification can be performed using a straightforward peak-guided strategy. Low priorities are assigned to extracts when activity cannot be recovered after fractionation or appears dispersed over a broad time window. The scale-up of the separation is straightforward, since the chromatographic system can be easily transposed to preparative HPLC. Examples of HPLC-based activity profiling in the context of industrial HTS programs have been recently reported. The application of this approach in a screening program for novel glucagon receptor antagonists demonstrates its potential for early dereplication. In this project, an extract of Streptomyces sp. strongly inhibited glucagon induced cAMP elevation. Semipreparative gradient HPLC separation of 30 µl of extract into 30 one-minute fractions and subsequent testing of each fraction enabled the activity to be attributed to a main peak at 16.1 min. (Fig. 5). HPLC-UV-MS analysis revealed a UV-spectrum similar to that of tryptophan and a MW of 2036 amu. Since no record corresponding to these data could be found in the literature, purification of the compound was undertaken on a preparative scale. The compound, a new bicyclic peptide (7), exhibited potent and selective antagonist activity towards the human glucagon receptor in a functional assay (IC50 0.44 µM) [51]. At the same time, micro-scale fractionation reliably filtered out extracts in which the inhibitory activity could be assigned to known compounds of little interest, such as cephalochromin (8) or resistomycin (9). The same strategy was successfully applied to the detection and isolation of a series of terphenylquinones as part of a HTS-supported lead finding process where more than 80’000 natural product extracts of plant and microbial origin were tested. Among other hits, an extract of a culture of the fungus Stillbella sp. showed a remarkable inhibition of the tyrosine kinase src, the prototype member of the src family of kinases involved in several signaling pathways. Comparative analysis of the LC-MS-UV dataset and the bioactivity plot of the fractionated extract indicated that the activity of this extract was based on a group of hydroxyquinone derivatives (10-13). Targeted purification of the active compounds was subsequently achieved by mass spectrometry-controlled preparative HPLC using ESIMS. The most potent compound, the trihydroxyphenyl derivative 12, inhibits the tyrosine kinase activity of human src with an IC50 of 3.9 µM [52]. A similar approach was also applied in the course of a screening program for new CDK5 inhibitors. CDK5 is a cyclin dependent kinase playing a crucial role in the development of the central nervous system and the regulation of neuronal signal transduction. A lipophilic extract of the stems and leaves of the shrub Clausena excavata Burm. (Rutaceae) was selected upon HPLC-based activity profiling. Activity profile and spectroscopic data strongly pointed towards a compound with a UV spectrum characteristic of a 9H-carbazole chromophor. Peak-guided isolation afforded a new alkaloid (14) with potent antagonist activity (IC50 0.51 µM) [53]. 15,16-dihydrotanshinone I (4) cryptotanshinone (5) tanshinone I (6) O O O O O O O O O O O O tanshinone IIA (3)