Predicting crystal form stability under real-world conditions.

The physicochemical properties of molecular crystals, such as solubility, stability, compactability, melting behaviour and bioavailability, depend on their crystal form1. In silico crystal form selection has recently come much closer to realization because of the development of accurate and affordable free-energy calculations2-4. Here we redefine the state of the art, primarily by improving the accuracy of free-energy calculations, constructing a reliable experimental benchmark for solid-solid free-energy differences, quantifying statistical errors for the computed free energies and placing both hydrate crystal structures of different stoichiometries and anhydrate crystal structures on the same energy landscape, with defined error bars, as a function of temperature and relative humidity. The calculated free energies have standard errors of 1-2 kJ mol-1 for industrially relevant compounds, and the method to place crystal structures with different hydrate stoichiometries on the same energy landscape can be extended to other multi-component systems, including solvates. These contributions reduce the gap between the needs of the experimentalist and the capabilities of modern computational tools, transforming crystal structure prediction into a more reliable and actionable procedure that can be used in combination with experimental evidence to direct crystal form selection and establish control5.

cis-Selective synthesis of 1,3-disubstituted tetrahydro-ß-carbolines from N-sulfonyl N,S-acetals.

Nucleophilic addition of Grignard reagents to tetrahydro-ß-carboline (THC) N-sulfonyl N,S-acetal generates exclusively cis-1,3-disubstituted THCs with a unique 1,3-diaxial conformation. The stereochemical relationship of the 1,3-substituents was confirmed by 2-dimensional NMR spectroscopy and X-ray crystallography. The mechanism of the reaction is proposed based on crystal structures and molecular orbital calculations.

Understanding Phase Behavior of Nearly Energetically Equivalent Polymorphs To Achieve Controlled Crystallization for a Nav1.7 Pain Inhibitor Compound.

GENE-A, a Nav1.7 inhibitor compound with analgesic activity, was developed as a crystalline anhydrate, for which two polymorphic forms, I and II, were discovered. The two forms were found to possess very similar free energies as determined experimentally with Form II being thermodynamically stable above 25 °C based on solubility measurements. A detailed solid-state characterization was conducted to determine the relative stability of these solid forms, and both thermodynamic and kinetic pathways (slurry bridging and crystallization) were evaluated. Form II was obtained as the final form in competitive slurries at RT. The outcome of crystallization experiments in terms of the solid form obtained was complicated and yielded variable results depending on the form of the starting material and that of the seeds. Form II was reproducibly obtained as the end product in unseeded experiments and in those with Form II as seeds and starting material, while Form I was obtained in all other seeded experiments. On the basis of the experimental data, a controlled crystallization strategy was developed, wherein Form II was used both as starting material and seeds to reproducibly obtain the desired form upon scale-up.

Phosphonium Formation by Facile Carbon-Phosphorus Reductive Elimination from Gold(III).

A recent trend in homogeneous gold catalysis has been the development of oxidative transformations relying on Au(I)/Au(III) redox cycling. Typically, phosphine-supported Au(I) precatalysts are used in the presence of strong oxidants to presumably generate phosphine Au(III) intermediates. Herein, we disclose that such Au(III) complexes can undergo facile C(aryl)-P reductive elimination to afford phosphonium salts, which have been spectroscopically and crystallographically characterized. Mechanistic studies indicate that this process occurs from cationic species at temperatures as low as -20 °C but can be accelerated in the presence of nucleophiles, such as acetonitrile and phosphines, via a five-coordinate intermediate. Importantly, this study highlights that irreversible C(aryl)-P reductive elimination is a feasible decomposition or activation pathway for phosphine-supported Au(III) catalysts and should not be ignored in future reaction development.

Syntheses of Denudatine Diterpenoid Alkaloids: Cochlearenine, N-Ethyl-1α-hydroxy-17-veratroyldictyzine, and Paniculamine.

The denudatine-type diterpenoid alkaloids cochlearenine, N-ethyl-1α-hydroxy-17-veratroyldictyzine, and paniculamine have been synthesized for the first time (25, 26, and 26 steps from 16, respectively). These syntheses take advantage of a common intermediate (8) that we have previously employed in preparing aconitine-type natural products. The syntheses reported herein complete the realization of a unified strategy for the preparation of C20, C19, and C18 diterpenoid alkaloids.

Napyradiomycin derivatives, produced by a marine-derived actinomycete, illustrate cytotoxicity by induction of apoptosis.

The microbial production, isolation, and structure elucidation of four new napyradiomycin congeners (1-4) is reported. The structures of these compounds, which are new additions to the marine-derived meroterpenoids, were defined by comprehensive spectroscopic analysis and by X-ray crystallography. Using fluorescence-activated cell sorting (FACS) analysis, napyradiomycins 1-4 were observed to induce apoptosis in the colon adenocarcinoma cell line HCT-116, indicating the possibility of a specific biochemical target for this class of cytotoxins.

Chiral amide directed assembly of a diastereo- and enantiopure supramolecular host and its application to enantioselective catalysis of neutral substrates.

The synthesis of a novel supramolecular tetrahedral assembly of K12Ga4L6 stoichiometry is reported. The newly designed chiral ligand exhibits high diastereoselective control during cluster formation, leading exclusively to a single diastereomer of the desired host. This new assembly also exhibits high stability toward oxidation or a low pH environment and is a more robust and efficient catalyst for asymmetric organic transformations of neutral substrates.

Effect of basic site substituents on concerted proton-electron transfer in hydrogen-bonded pyridyl-phenols.

Separated concerted proton-electron transfer (sCPET) reactions of two series of phenols with pendent substituted pyridyl moieties are described. The pyridine is either attached directly to the phenol (HOAr-pyX) or connected through a methylene linker (HOArCH(2)pyX) (X = 4-NO(2), 5-CF(3), 4-CH(3), and 4-NMe(2)). Electron-donating and -withdrawing substituents have a substantial effect on the chemical environment of the transferring proton, as indicated by IR and (1)H NMR spectra, X-ray structures, and computational studies. One-electron oxidation of the phenols occurs concomitantly with proton transfer from the phenolic oxygen to the pyridyl nitrogen. The oxidation potentials vary linearly with the pK(a) of the free pyridine (pyX), with slopes slightly below the Nerstian value of 59 mV/pK(a). For the HOArCH(2)pyX series, the rate constants k(sCPET) for oxidation by NAr(3)(•+) or [Fe(diimine)(3)](3+) vary primarily with the thermodynamic driving force (ΔG°(sCPET)), whether ΔG° is changed by varying the potential of the oxidant or the substituent on the pyridine, indicating a constant intrinsic barrier λ. In contrast, the substituents in the HOAr-pyX series affect λ as well as ΔG°(sCPET), and compounds with electron-withdrawing substituents have significantly lower reactivity. The relationship between the structural and spectroscopic properties of the phenols and their CPET reactivity is discussed.

Challenges in the structure determination of a dimeric impurity found during development of GDC-0326.

While developing a synthetic route for GDC-0326, a PI3Kα selective inhibitor, a side product was identified which was adversely impacting process chemistry development. To aid in optimization of a viable synthetic pathway for the drug, it was decided to characterize this impurity. Initial efforts using typical high-resolution mass spectrometry data coupled with NMR analysis were unable to unambiguously identify the structure. The NMR analysis was hampered by a severe lack of protons in the core of the structure. While efforts were being made to produce suitable crystals for definitive x-ray analysis, Raman analysis was undertaken. The vibrational data were compared to DFT calculations for the two most likely structures. This data, along with chemical reasoning, eventually led to successful prediction of structure 2, which was ultimately confirmed by single crystal x-ray diffractometry data.

Intramolecular and intermolecular contributions to the barriers for rotation of methyl groups in crystalline solids: electronic structure calculations and solid-state NMR relaxation measurements.

The rotation barriers for 10 different methyl groups in five methyl-substituted phenanthrenes and three methyl-substituted naphthalenes were determined by ab initio electronic structure calculations, both for the isolated molecules and for the central molecules in clusters containing 8-13 molecules. These clusters were constructed computationally using the carbon positions obtained from the crystal structures of the eight compounds and the hydrogen positions obtained from electronic structure calculations. The calculated methyl rotation barriers in the clusters (E(clust)) range from 0.6 to 3.4 kcal/mol. Solid-state (1)H NMR spin-lattice relaxation rate measurements on the polycrystalline solids gave experimental activation energies (E(NMR)) for methyl rotation in the range from 0.4 to 3.2 kcal/mol. The energy differences E(clust) - E(NMR) for each of the ten methyl groups range from -0.2 kcal/mol to +0.7 kcal/mol, with a mean value of +0.2 kcal/mol and a standard deviation of 0.3 kcal/mol. The differences between each of the computed barriers in the clusters (E(clust)) and the corresponding computed barriers in the isolated molecules (E(isol)) provide an estimate of the intermolecular contributions to the rotation barriers in the clusters. The values of E(clust) - E(isol) range from 0.0 to 1.0 kcal/mol.

Synthesis with structural and electronic characterization of homoleptic Fe(II)- and Fe(III)-fluorinated phenolate complexes.

Four Fe(III) compounds and one Fe(II) compound containing mononuclear, homoleptic, fluorinated phenolate anions of the form [Fe(OAr)(m)](n-) have been prepared in which Ar(F) = C(6)F(5) and Ar' = 3,5-C(6)(CF(3))(2)H(3): (Ph(4)P)(2)[Fe(OAr(F))(5)], 1, (Me(4)N)(2)[Fe(OAr(F))(5)], 2, {K(18-crown-6)}(2)[Fe(OAr(F))(5)], 3a, {K(18-crown-6)}(2)[Fe(OAr')(5)], 3b, and {K(18-crown-6)}(2)[Fe(OAr(F))(4)], 6. Two dinuclear Fe(III) compounds have also been prepared: {K(18-crown-6)}(2)[(OAr(F))(3)Fe(µ(2)-O)Fe(OAr(F))(3)], 4, and {K(18-crown-6)}(2)[(OAr(F))(3)Fe(µ(2)-OAr(F))(2)Fe(OAr(F))(3)], 5. These compounds have been characterized with UV-vis spectroscopy, elemental analysis, Evans method susceptibility, and X-ray crystallography. All-electron, geometry-optimized DFT calculations on four [Ti(IV)(OAr)(4)] and four [Fe(III)(OAr)(4)](-) species (Ar = 2,3,5,6-C(6)Me(4)H, C(6)H(5), 2,4,6-C(6)Cl(3)H(2), C(6)F(5)) with GGA-BP and hybrid B3LYP basis sets demonstrated that, under D(2d) symmetry, π donation from the O 2p orbitals is primarily into the d(xy) and d(z(2)) orbitals. The degree of donation is qualitatively consistent with expectations based on ligand Brønsted basicity and supports the contention that fluorinated phenolate ligands facilitate isolation of nonbridged homoleptic complexes due to their reduced π basicity at oxygen.

Probing concerted proton-electron transfer in phenol-imidazoles.

A series of seven substituted 4,6-di-tert-butyl-2-(4,5-diarylimidazolyl)-phenols have been prepared and characterized, along with two related benzimidazole compounds. X-ray crystal structures of all of the compounds show that the phenol and imidazole rings are close to coplanar and are connected by an intramolecular ArOHN hydrogen bond. One-electron oxidation of these compounds occurs with movement of the phenolic proton to the imidazole base by concerted proton-electron transfer (CPET) to yield fairly stable distonic radical cations. These phenol-base compounds are a valuable system in which to examine the key features of CPET. Kinetic measurements of bimolecular CPET oxidations, with E(rxn) between +0.04 and -0.33 V, give rate constants from (6.3 +/- 0.6) x 10(2) to (3.0 +/- 0.6) x 10(6) M(-1) s(-1). There is a good correlation of log(k) with DeltaG degrees , with only one of the 15 rate constants falling more than a factor of 5.2 from the correlation line. Substituents on the imidazole affect the (O-HN) hydrogen bond, as marked by variations in the (1)H NMR and calculated vibrational spectra and geometries. Crystallographic d(ON) values appear to be more strongly affected by crystal packing forces. However, there is almost no correlation of rate constants with any of these measured or computed parameters. Over this range of compounds from the same structural family, the dominant contributor to the differences in rate constant is the driving force DeltaG degrees .

Two crystallographic forms and the absolute structure of 5α,14α-androstane.

5α,14α-Androstane (C19H32) crystallizes in two different polymorphic forms in the same vapor diffusion experiment. The major form (Form I) crystallizes as thin plates in the space group P21, with Z = 4. These plates are twinned along a long c axis of length 43 Šand readily suffer from radiation damage when diffracted. The minor form (Form II) crystallizes as fine needles in the space group P212121, Z = 3. In the minor form, 5α,14α-androstane cocrystallizes with 5α,14α-androstan-17-one, an oxidation product of 5α,14α-androstane. The presence of 5α,14α-androstan-17-one in the minor form of the crystals was confirmed by HR-MS. Form II can be crystallized as a pure form without the ketone impurity using a different solvent system. High level density functional theory (DFT) lattice free energy calculations were performed and show that both pure forms are isoergic within the estimated error of the calculations.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

GDC-9545 (Giredestrant): A Potent and Orally Bioavailable Selective Estrogen Receptor Antagonist and Degrader with an Exceptional Preclinical Profile for ER+ Breast Cancer.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Multimodal study of secondary interactions in Cp*Ir complexes of imidazolylphosphines bearing an NH group.

Hydrogen bonding phenomena are explored using a combination of X-ray diffraction, NMR and IR spectroscopy, and DFT calculations. Three imidazolylphosphines R(2)PImH (ImH = imidazol-2-yl, R = t-butyl, i-propyl, phenyl, 1a-1c) and control phosphine (i-Pr)(2)PhP (1d) lacking an imidazole were used to make a series of complexes of the form Cp*Ir(L(1))(L(2))(phosphine). In addition, in order to suppress intermolecular interactions with either imidazole nitrogen, 1e, a di(isopropyl)imidazolyl analogue of 1b was made along with its doubly (15)N-labeled isotopomer to explore bonding interactions at each imidazole nitrogen. A modest enhancement of transfer hydrogenation rate was seen when an imidazolylphosphine ligand 1b was used. Dichloro complexes (L(1) = L(2) = Cl, 2a-2c,2e) showed intramolecular hydrogen bonding as revealed by four X-ray structures and various NMR and IR data. Significantly, hydride chloride complexes [L(1) = H, L(2) = Cl, 3a-3c and 3e-((15)N)(2)] showed stronger hydrogen bonding to chloride than hydride, though the solid-state structure of 3b evinced intramolecular Ir-H...H-N bonding reinforced by intermolecular N...H-N bonding between unhindered imidazoles. These results are compared to literature examples, which show variations in preferred hydrogen bonding to hydride, halide, CO, and NO ligands. Surprising differences were seen between the dichloro complex 2b with isopropyl groups on phosphorus, which appeared to exist as a mixture of two conformers, and related complex 2a with tert-butyl groups on phosphorus, which exists in chlorinated solvents as a mixture of conformer 2a-endo and chelate 5a-Cl, the product of ionization of one chloride ligand. This difference became apparent only through a series of experiments, especially (15)N chemical shift data from 2D (1)H-(15)N correlation. The results highlight the difficulty of characterizing hemilabile, bifunctional complexes and the importance of innocent ligand substituents in determining structure and dynamics.

Synthesis and reactivity of oxo-peroxo-vanadium(V) bipyridine compounds.

The vanadium(IV) compound [V(IV)O(OH)((t)Bu(2)bpy)(2)]BF(4) (V(IV)O(OH)) ((t)Bu(2)bpy = 4,4'-di-tert-butylbipyridine) is slowly oxidized by O(2) in ethereal solvents to give the oxo-peroxo compound [V(V)O(O(2))((t)Bu(2)bpy)(2)]BF(4) (V(V)O(O(2))) in excellent yield. This and related compounds were fully characterized by NMR, IR, and optical spectroscopies; mass spectrometry; elemental analyses; and an X-ray crystal structure of the 4,4'-dimethylbipyridine analog, [V(V)O(O(2))(Me(2)bpy)(2)]BF(4). Monitoring the reaction of V(IV)O(OH) with O(2) in THF/acetonitrile mixtures by (1)H NMR and optical spectroscopies surprisingly shows that the initial product is the cis-dioxo compound [V(V)(O)(2)((t)Bu(2)bpy)(2)]BF(4) (V(V)O(2)), which then converts to V(V)O(O(2)). Reaction of V(IV)O(OH) with (18)O(2) gives ca. 60% triply (18)O labeled V(V)O(O(2)). The mechanism of formation of V(V)O(O(2)) is complex and may occur via initial reduction of O(2) at vanadium(IV) to give a superoxo-vanadium(V) intermediate, autoxidation of the THF solvent, or both. That V(V)O(2) is generated first appears to be due to the ability of V(IV)O(OH) to act as a hydrogen atom donor. For instance, V(IV)O(OH) reacts with V(V)O(O(2)) to give V(V)O(2). V(V)O(O(2)) is also slowly reduced to V(IV)O(OH) by the organic hydrogen atom donors hydroquinone and TEMPOH (2,2,6,6-tetramethylpiperidin-1-ol) as well as by triphenylphosphine. Notably, the peroxo complex V(V)O(O(2)) is much less reactive with these substrates than the analogous dioxo compound V(V)O(2).

Uranium metallocene complexes of the 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidinato ligand, (hpp)(-).

The utility of the 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidinato ligand, (hpp)(-), in uranium chemistry has been probed by synthesizing metallocene complexes and studying their reactivity. (C(5)Me(5))(2)UMe(2) reacts with 1 equiv of Hhpp to form (C(5)Me(5))(2)(hpp)UMe, 1, which does not react further with Hhpp. (C(5)Me(5))(2)UCl(2) reacts with Khpp to form (C(5)Me(5))(2)(hpp)UCl, 2, which similarly does not react with additional Khpp. Complex 2 reacts with NaN(3) to form the azide complex, (C(5)Me(5))(2)(hpp)UN(3), 3. The trivalent uranium (hpp)(-) metallocene complex, (C(5)Me(5))(2)(hpp)U, 4, can be synthesized by the reaction of [(C(5)Me(5))(2)U][BPh(4)] with Khpp and from 2 with KC(8). Complex 4 can be oxidized with Ph(3)P horizontal lineSe to produce the tetravalent product, [(C(5)Me(5))(2)(hpp)U](2)(mu-Se), 5. The reaction of 4 with Me(3)SiN(3) provides the pentavalent uranium complex, (C(5)Me(5))(2)(hpp)U(=NSiMe(3)), 6.

Observation of redox-induced electron transfer and spin crossover for dinuclear cobalt and iron complexes with the 2,5-di-tert-butyl-3,6-dihydroxy-1,4-benzoquinonate bridging ligand.

Dinuclear [(TPyA)M(II)(DBQ(2-))M(II)(TPyA)](BF(4))(2) [TPyA = tris(2-pyridylmethyl)amine; DBQ(2-) = 2,5-di-tert-butyl-3,6-dihydroxy-1,4-benzoquinonate; M = Co (1(2+)), Fe (2(2+)), Ni (3(2+))] complexes have been prepared by the reaction of M(2+), TPyA, H(2)DBQ, and triethylamine in MeOH solution. Their monooxidized form [(TPyA)M(III)(DBQ(*3-))M(III)(TPyA)](3+) [Co = (1(3+)), Fe (2(3+))] has been synthesized by using ferrocenium tetrafluoroborate, and the dioxidized form of 1(2+), [(TPyA)Co(III)(DBQ(2-))Co(III)(TPyA)](4+) (1(4+)), has been obtained by using thianthrinium tetrafluoroborate. These dinuclear compounds were characterized by X-ray crystallography, electrochemistry, magnetism, and EPR spectroscopy. Valence ambiguous 1(3+) forms via redox-induced electron transfer, whereby the one-electron oxidation of the [Co(II)(DBQ(2-))Co(II)](2+) core forms [Co(III)(DBQ(*3-))Co(III)](3+), and it also exhibits spin crossover behavior to the core [Co(III)(DBQ(2-))Co(II)](3+) above room temperature. The M ions in 1 and 2 have a distorted octahedral geometry by coordination with four nitrogens of a TPyA, two oxygens of a DBQ(2-/*3-). Due to the interdimer offset face-to-face pi-pi and/or herringbone interactions, 1(2+), 1(3+), and 2(2+) show extended 1-D and/or 2-D supramolecular structures. The existence of DBQ(*3-) in 1(3+) is confirmed from both solid-state magnetic and solution EPR data. Co- and Ni-based 1(2+) and 3(2+) show weak antiferromagnetic interactions [1(2+): g = 2.44, J/k(B) = -3.20 K (-2.22 cm(-1)); 3(2+): g = 2.13, J/k(B) = -3.22 K (-2.24 cm(-1)), H = -2JS(1)*S(2) for 1(2+) and 3(2+)], while Fe-based 2(2+) exhibits strong spin crossover behavior above room temperature. 1(2+) has three reversible one-electron transfer waves at E(1/2) (vs SCE in MeCN) = -1.121, 0.007, and 0.329 V, and a fourth wave at -1.741 V that exhibits a slight chemical irreversibility. The first three correspond to [Co(II)DBQ(2-)Co(II)](2+) reduction to [Co(II)DBQ(*3-)Co(II)](+), and oxidation to [Co(III)DBQ(*3-)Co(III)](3+) and [Co(III)DBQ(2-)Co(III)](4+), respectively. The mechanism of the multielectron transfer oxidation from [Co(II)DBQ(2-)Co(II)](2+) to [Co(III)DBQ(*3-)Co(III)](3+) is unknown; the energy of stabilization for oxidizing the Co(II) centers in the presence of DBQ(*3-), relative to oxidizing the Co(II) centers in the presence of DBQ(2-) is computed to be 1.45 eV. 2(2+) also has three reversible one-electron transfer waves at 0.802, 0.281, and -1.007 V that correspond to two successive one-electron oxidations (2(2+)/2(3+) and 2(3+)/2(4+)), and a one-electron reduction (2(2+)/2(+)). 2(2+) has the [Fe(hs)(II)(DBQ(2-))Fe(hs)(II)](2+) electronic structure that becomes [Fe(hs)(III)(DBQ(*3-))Fe(hs)(III)](3+) upon oxidation. The latter undergoes spin crossover above room temperature to populate the [Fe(hs)(III)(DBQ(2-))Fe(hs)(II)](3+) excited state.

Isolation, structure elucidation, and antitumor activity of spirohexenolides A and B.

In this report, we describe the discovery of a pair of bioactive spirotetronates, spirohexenolides A (1) and B (2), that arose from the application of mutagenesis, clonal selection techniques, and media optimization to strains of Streptomyces platensis. The structures of spirohexenolides A (1) and B (2) were elucidated through X-ray crystallography and confirmed by 1D and 2D NMR studies. Under all examined culture conditions, spirohexenolide A (1) was the major metabolite with traces of spirohexenolide B (2) arising in cultures containing increased loads of adsorbent resins. Spirohexenolide A (1) inhibited tumor cell growth with GI(50) values spanning from 0.1 to 17 microM across the NCI 60 cell line panel. An increased activity was observed in leukemia (GI(50) value of 254 nM in RPMI-8226 cells), lung cancer (GI(50) value of 191 nM in HOP-92 cells), and colon cancer (GI(50) value of 565 nM in SW-620 cells) tumor cells. Metabolite 1 was fluorescent and could be examined on a confocal fluorescent microscope with conventional laser excitation and filter sets. Time lapse imaging studies indicated that spirohexenolide A (1) was readily taken up by tumor cells, appearing through the cell immediately after dosing and subcellularly localizing in the lysosomes. This activity, combined with a unique selectivity in NCI 60 cancer cell line screening, indicates that 1 warrants further chemotherapeutic evaluation.