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The strongest mechanism for adsorption of antigens to aluminum adjuvants is ligand exchange, which involves the replacement of a surface hydroxyl on the adjuvant by a terminal phosphate group of the antigen. A novel phosphonate linker was developed that allows the addition of phosphonate (C-PO3) groups to proteins under controlled and chemically mild conditions. Increasing the number of linkers per protein molecule progressively increased the adsorption strength to aluminum hydroxide adjuvant (AH) as measured by elution in serum. The effect of phosphonate conjugation on the antibody response was determined with hen egg lysozyme (HEL), a protein that has the same charge as AH at neutral pH and does not adsorb to AH. The phosphonylated form of HEL (HEL-P) adsorbed to AH, indicating that the ligand exchange interaction could overcome the electrostatic repulsion. Mice injected with HEL-P/AH had a higher antibody titer to HEL than mice injected with HEL/AH, especially at lower antigen doses, suggesting that adsorption of antigen has a dose-sparing effect. Conjugation of CRM197, an antigen that adsorbs electrostatically to AH, with phosphonate linkers did not enhance the antibody response, indicating that adsorption by either electrostatic or ligand exchange to AH is sufficient to enhance the antibody response.

SH2 domains are attractive targets for chemotherapeutic agents due to their involvement in the formation of protein-protein interactions critical to many signal transduction cascades. Little is known, however, about how synthetic SH2 domain ligands would influence the growth properties of tumor cells or with which intracellular proteins they would interact due to their highly charged nature and enzymatic lability. In this study, a prodrug delivery strategy was used to introduce an enzymatically stable, phosphotyrosine peptidomimetic into tumor cells. When tested in a human tumor cell panel, the prodrug exhibited a preference for inhibiting the growth of leukemia and lymphoma cells. In these cells, it was largely cytostatic and induced endoreduplication and the appearance of midbodies. Proteomic analyses identified multiple targets that included mitotic centromere-associated kinesin (MCAK). Molecular modeling studies suggested the ATP-binding site on MCAK as the likely site of drug interaction. Consistent with this, ATP inhibited the drug-MCAK interaction and the drug inhibited MCAK ATPase activity. Accordingly, the effects of the prodrug on the assembly of the mitotic spindle and alignment of chromosomes were consistent with the identification of MCAK as an important intracellular target.

Summary Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues. To create small molecules that inhibit such interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. For this study, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The prodrug is cytotoxic at low micromolar concentrations when applied to cancer cells and induces caspase activation resulting in apoptosis. The prodrug reverses the 14-3-3-mediated inhibition of FOXO3a resulting from its phosphorylation by Akt1 in a concentration-dependent manner that correlates well with its ability to inhibit cell growth. This methodology can be applied to target a variety of proteins containing phosphoserine and other phosphoamino acid binding domains.

APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC50 values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development. Antioxid. Redox Signal. 14, 1387–1401.

Prenylation inhibitors have gained increasing attention as potential therapeutics for cancer. Initial work focused on inhibitors of farnesylation, but more recently geranylgeranyl transferase inhibitors (GGTIs) have begun to be evaluated for their potential antitumor activity in vitro and in vivo. In this study, we have developed a nonpeptidomimetic GGTI, termed GGTI-2Z [(5-nitrofuran-2-yl)methyl-(2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl 4-chlorobutyl(methyl)phosphoramidate], which in combination with lovastatin inhibits geranylgeranyl transferase I (GGTase I) and GGTase II/RabGGTase, without affecting farnesylation. The combination treatment results in a G0/G1 arrest and synergistic inhibition of proliferation of cultured STS-26T malignant peripheral nerve sheath tumor cells. We also show that the antiproliferative activity of drugs in combination occurs in the context of autophagy. The combination treatment also induces autophagy in the MCF10.DCIS model of human breast ductal carcinoma in situ and in 1c1c7 murine hepatoma cells, where it also reduces proliferation. At the same time, there is no detectable toxicity in normal immortalized Schwann cells. These studies establish GGTI-2Z as a novel geranylgeranyl pyrophosphate derivative that may work through a new mechanism involving the induction of autophagy and, in combination with lovastatin, may serve as a valuable paradigm for developing more effective strategies in this class of antitumor therapeutics.

Neurofibromatosis type 1 (NF1) is a genetic disorder that is driven by the loss of neurofibromin (Nf) protein function. Nf contains a Ras-GTPase-activating protein domain, which directly regulates Ras signaling. Numerous clinical manifestations are associated with the loss of Nf and increased Ras activity. Ras proteins must be prenylated to traffic and functionally localize with target membranes. Hence, Ras is a potential therapeutic target for treating NF1. We have tested the efficacy of two novel farnesyl transferase inhibitors (FTIs), 1 and 2, alone or in combination with lovastatin, on two NF1 malignant peripheral nerve sheath tumor (MPNST) cell lines, NF90-8 and ST88-14. Single treatments of 1, 2, or lovastatin had no effect on Ras prenylation or MPNST cell proliferation. However, low micromolar combinations of 1 or 2 with lovastatin (FTI/lovastatin) reduced Ras prenylation in both MPNST cell lines. Furthermore, this FTI/lovastatin combination treatment reduced cell proliferation and induced an apoptotic response as shown by morphological analysis, procaspase-3/-7 activation, loss of mitochondrial membrane potential, and accumulation of cells with sub-G(1) DNA content. Little to no detectable toxicity was observed in normal rat Schwann cells following FTI/lovastatin combination treatment. These data support the hypothesis that combination FTI plus lovastatin therapy may be a potential treatment for NF1 MPNSTs.

Our laboratory recently reported the development of novel prodrug chemistry for the intracellular delivery of phosphotyrosine mimetics. This chemistry has now been adapted for the synthesis of a prodrug that delivers the nonhydrolyzable difluoromethylphosphonate moiety intracellularly. Activation of the prodrug generates a difluoromethylphosphonamidate anion that undergoes subsequent cyclization and hydrolysis with a t1/2 = 44 min. A highly potent and selective inhibitor of protein tyrosine phosphatase 1B (PTP1B) with a nanomolar Ki has been reported, but this bis(difluoromethylphosphonate) lacks potential utility due to its exceedingly low membrane permeability at physiological pH. A prodrug of this inhibitor has been synthesized and evaluated in a cell-based assay. The prodrug exhibits nanomolar PTP1B inhibitory activity in this assay, confirming the efficacy of intracellular phosphonate delivery using this prodrug approach.

Both enantiomers of protected and unprotected 2-hydroxymethylaziridines are efficiently and enantiospecifically synthesized by using a combination of enzymatic and synthetic methods. PPL was used for lipase-catalyzed desymmetrization of N-protected serinol. [reaction: see text].

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome. NF1 patients present with a constellation of clinical manifestations and have an increased risk of developing certain benign and malignant tumors. This disease results from mutation within the gene encoding neurofibromin, a GTPase activating protein (GAP) for Ras. Functional loss of this protein compromises Ras inactivation, which leads to the aberrant growth and proliferation of neural crest-derived cells and, ultimately, tumor formation. Current management of NF1-associated malignancy involves radiation, surgical excision, and cytotoxic drugs. The limited success of these strategies has fueled researchers to further elucidate the molecular changes that drive tumor formation and progression. This discussion will highlight how intracellular signaling molecules, cell-surface receptors, and the tumor microenvironment constitute potential therapeutic targets, which may be relevant not only to NF1-related malignancy but also to other human cancers.

A novel approach to the intracellular delivery of aryl phosphates has been developed that utilizes a phosphoramidate-based prodrug approach. The prodrugs contain an ester group that undergoes reductive activation intracellularly with concomitant expulsion of a phosphoramidate anion. This anion undergoes intramolecular cyclization and hydrolysis to generate aryl phosphate exclusively with a t(1/2) = approximately 20 min. Phosphoramidate prodrugs (8-10) of phosphate-containing peptidomimetics that target the SH2 domain were synthesized. Evaluation of these peptidomimetic prodrugs in a growth inhibition assay and in a cell-based transcriptional assay demonstrated that the prodrugs had IC50 values in the low micromolar range. Synthesis of phosphorodiamidate analogues containing a P-NH-Ar linker (16-18) was also carried out in the hope that the phosphoramidates released might be phosphatase-resistant. Comparable activation rates and cell-based activities were observed for these prodrugs, but the intermediate phosphoramidate dianion underwent spontaneous hydrolysis with a t(1/2) = approximately 30 min.

Previously a series of 2- and 3-substituted indolequinone phosphoramidate prodrugs was synthesized, and the compounds were shown to be nanomolar inhibitors of cell proliferation. The activation of these compounds following both one- and two-electron reduction has been investigated. (31)P NMR experiments demonstrated that both series of compounds undergo rapid activation following two-electron reduction. Additionally, the 3-series of compounds undergo rapid activation following one-electron reduction, while activation of the 2-series of compounds via this mechanism is very slow. The activation of these prodrugs by direct displacement using sulfur nucleophiles such as glutathione has been examined. Activation via this route is rapid for the 3-regioisomers, but is considerably slower for the 2-substituted analogues under similar conditions. Together these findings suggest that drug delivery via two-electron reduction from the 2-position is the more selective prodrug strategy.

A series of 2- and 3-substituted indolequinone phosphoramidate prodrugs targeted to DT-diaphorase (DTD) have been synthesized and evaluated. These compounds are designed to undergo activation via quinone reduction by DTD followed by expulsion of the phosphoramide mustard substituent from the hydroquinone. Chemical reduction of the phosphoramidate prodrugs led to rapid expulsion of the corresponding phosphoramidate anions in both series of compounds. Compounds substituted at the 2-position are excellent substrates for human DTD (k(cat)/K(M) = (2-5) x 10(6) M(-1) s(-1)); however, compounds substituted at the 3-position are potent inhibitors of the target enzyme. Both series of compounds are toxic in HT-29 and BE human colon cancer cell lines in a clonogenic assay. There was a correlation found between cytotoxicity and DTD activity for the 2-series of phosphoramidates; however, there was no correlation between cytotoxicity and DTD activity in the 3-series of compounds. This finding suggests the presence of an alternative mechanism for the activation of these compounds.

[reaction--see text] Sugar nucleoside diphosphates have been prepared using an efficient phosphate coupling reaction that employs a highly reactive zwitterionic phosphoramidate intermediate as the phosphorylating species.

A series of naphthoquinone and benzimidazolequinone phosphorodiamidates has been synthesized and studied as potential cytotoxic prodrugs activated by DT-diaphorase. Reduction of the quinone moiety in the target compounds was expected to provide a pathway for expulsion of the phosphoramide mustard alkylating agent. All of the compounds synthesized were excellent substrates for purified human DT-diaphorase (k(cat)/K(m) = 3 x 10(7) - 3 x 10(8) M(-1) s(-1)). The naphthoquinones were toxic to both HT-29 and BE human colon cancer cell lines in a clonogenic assay; however, cytotoxicity did not correlate with DT-diaphorase activity in these cell lines. The benzimidazolequinone analogues were 1-2 orders of magnitude less cytotoxic than the naphthoquinone analogues. Chemical reduction of the naphthoquinone led to rapid expulsion of the phosphorodiamidate anion; in contrast, the benzimidazole reduction product was stable. Michael addition of glutathione and other sulfur nucleophiles provides an alternate mechanism for activation of the naphthoquinone phosphorodiamidates, and this mechanism may contribute to the cytotoxicity of these compounds.

A series of novel nitroheterocyclic phosphoramidates has been prepared, and the cytotoxicity of these compounds has been evaluated in clonogenic assays against B16, wild-type and cyclophosphamide-resistant MCF-7, and HT-29 cells under aerobic conditions and HT-29 cells under hypoxic conditions. All compounds were comparable in toxicity to wild-type and resistant MCF-7 cells and were also selectively toxic to HT-29 cells under hypoxic conditions (selectivity ratios 1.7 to >20). Analogues lacking the nitro group were not cytotoxic. Electron-withdrawing substituents increased cytotoxicity under aerobic conditions and thereby decreased hypoxic selectivity. In contrast, an electron-donating substituent markedly decreased both aerobic and hypoxic cytotoxicity but enhanced hypoxic selectivity. Chemical reduction of the nitro group resulted in rapid expulsion of the cytotoxic phosphoramide mustard. The most potent of these compounds show significant cytotoxicity under both aerobic and hypoxic conditions.

Diethyldithiocarbamate (DDTC) exhibits chemoprotective effects via reduced myelosuppression in mice treated with various chemotherapeutic agents. The mechanism of DDTC-mediated chemoprotection is believed to involve the induction and release of several cytokines, including interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte colony-stimulating factor (G-CSF). In the present study the roles of IL-1beta and TNF-alpha in DDTC-mediated G-CSF induction were examined using human long-term bone marrow cultures (hLTBMCs). Administration of IL-1 receptor antagonist (IL-1ra) to DDTC-treated hLTBMCs obviated the G-CSF induction profile and blocked the resultant colony proliferation, indicating that IL-1beta mediates DDTC-induced G-CSF release and hematopoiesis. IL-1beta mRNA levels were increased threefold over control following DDTC treatment of hLTBMCs, implying that DDTC induces IL-1beta at the level of transcription. Conversely, studies involving inhibition of DDTC-induced TNF-alpha synthesis, with the inhibitor MNX 160, had no effect on DDTC-induced G-CSF release or colony proliferation. These findings taken together strongly suggest that IL-1beta mediates the chemoprotective effects of DDTC.

A series of alkylating phosphoramidate analogs of 5-fluoro-2'-deoxyuridine has been prepared and their growth inhibitory activity evaluated against murine L1210 leukemia and B16 melanoma cells in vitro. These compounds were designed to undergo intracellular release of the phosphoramidate anions, which it was hoped would function as irreversible inhibitors of thymidylate synthase. The expectation was that binding of the nucleoside moiety would be followed by alkylation of the enzyme via the phosphoramidate. The chloride, bromide, iodide, and tosylate analogs were highly potent inhibitors of L1210 cell proliferation, with increased inhibition observed at both higher drug concentrations and longer exposure times. Addition of thymidine completely reversed the inhibition for all compounds, suggesting that these compounds are acting via inhibition of thymidylate synthase. Although the nonalkylating morpholine analog 1f was ca. 50-fold less potent than the methyl(chloroethyl)amino compound, the piperidine analog 1g was only 2-fold less potent, confirming that nitrogen basicity may be as important as the presence of an alkylating group. Addition of thymidine reversed the growth inhibition of the morpholine and piperidine analogs, suggesting that these compounds may also undergo intracellular conversion to 5-fluoro-2'-deoxyuridine 5'monophosphate. The thymidine and deoxyuridine derivatives 2 and 3 showed minimal growth inhibition in the L1210 assay. The alkylating analogs showed modest cytotoxicity against B16 melanoma cells, and the potency of the analogs was more dependent upon the alkylating moiety than on the 5-substituent.

Interleukin-1 (IL-1) treatment of human WI-38 lung fibroblasts results in granulocyte-macrophage colony-stimulating factor (GM-CSF) expression as well as a delayed increase in prostaglandin E2 (PGE2) production that closely correlates with the decline of GM-CSF mRNA levels. Pretreatment with PGE2 reduces the IL-1 induced GM-CSF mRNA and protein expression to 10% to 15% of control values at concentrations of PGE2 that are endogenously produced after IL-1 stimulation. Inhibition of PGE2 synthesis by indomethacin prolongs the IL-1 induced GM-CSF mRNA expression and increases the cumulative GM-CSF protein secretion. Exposure of WI-38 fibroblasts to PGE2 results in an increase in intracellular cyclic adenosine monophosphate (cAMP) levels. The inhibition of GM-CSF expression by PGE2 can be mimicked by stable cAMP analogs as well as cAMP elevating agents such as cholera toxin, forskolin, and isobutylmethylxanthine. Thus the inhibition exerted by PGE2 is mediated via cAMP. Taken together, these results suggest that IL-1 stimulation of human fibroblasts provides not only the upregulatory signal for GM-CSF expression but also a delayed and indirect downregulatory signal that serves to limit GM-CSF expression in the continued presence of IL-1.

A series of novel nitrobenzyltetrakis(chloroethyl)phosphorodiamidates has been prepared, and its cytotoxicity has been evaluated against HT-29 cells under aerobic and hypoxic conditions and against murine bone marrow progenitor cells under aerobic conditions. All compounds were selectively toxic to HT-29 cells under hypoxic conditions, and the selectivity ratios varied from 1.6 to > 90. Analogs lacking either the nitro group or the tetrakis(chloroethyl) moiety were not cytotoxic, confirming that the presence of both nitro and incipient alkylating groups are essential for activity. Surprisingly, some analogs were far more toxic to bone marrow progenitors than to HT-29 cells under aerobic conditions, suggesting that other activation mechanisms must exist in these hematopoietic cells. Cytotoxicity increased with increasing depth in the HT-29 spheroid model, consistent with the preferential hypoxic toxicity of these compounds. Alkaline elution experiments showed a greater number of DNA interstrand cross-links under hypoxic compared to aerobic conditions. The extent of cross-linking in hypoxic cells was essentially identical to that produced by an equitoxic dose of melphalan, suggesting that the cytotoxicity of these compounds results from phosphorodiamidate release and alkylation of DNA.

Synthesis of a potential Src family SH2 domain inhibitor incorporating a 1,4-cis-enediol scaffold is reported. The synthetic route offers straightforward and highly selective access to the enediol and its associated chiral centers. Key steps include stereocontrolled syn-aldol coupling, amide alkynylation, and asymmetric ketone reduction.

A series of perhydrooxazine analogues of aldophosphamide has been prepared, and their 31P NMR kinetics and in vitro cytotoxicity have been evaluated. These compounds were developed on the basis of the idea that ring opening and tautomerization to an enamine intermediate might provide a mechanistic alternative to the beta-elimination reaction for release of phosphoramide mustard. The 4,4,6-trimethyltetrahydro-1,3-oxazine moiety was selected on the basis of its rapid rate of iminium ion generation and relatively slow rate of hydrolysis. These analogues underwent phosphorodiamidate release by three distinct mechanisms: hydrolysis to aldophosphamide and subsequent beta-elimination; cyclization to produce the 4-hydroxycyclophosphamides, which release phosphorodiamidate by ring opening and elimination; and tautomerization to the enamine with rapid expulsion of phosphorodiamidate. Kinetic studies demonstrated that hydrolysis to the aldehyde contributed minimally to the overall activation process and that the enamine pathway represented the major route of activation. For those analogues that could undergo cyclization, this pathway competed effectively with enamine release, and these analogues were essentially equivalent to their 4-hydroxycyclophosphamide counterparts in cytotoxicity. A series of tetra-N-substituted phosphorodiamidates that cannot undergo cyclization was prepared to explore the effects of cyclization on the cytotoxicity of these analogues. The tetrakis(chloroethyl)phosphorodiamidates were highly potent in vitro against both cyclophosphamide-sensitive and -resistant L1210 and P388 cell lines, and one of these analogues had significant antitumor activity against L1210 leukemia in vivo. These results demonstrate that the enamine mechanism provides a viable pathway for delivery of phosphorodiamidates and that this approach can be used to deliver phosphorodiamidates that are non-cross-resistant in cyclophosphamide-resistant cell lines.

A series of 5- and 6-substituted cyclophosphamide analogues has been prepared, and their 31P NMR kinetics of phosphoramide mustard (PDA) release and in vitro and in vivo cytotoxicity have been evaluated. cis-4-Hydroxy-5-methoxycyclophosphamide equilibrated very slowly and to a minor extent with the ring-opened aldophosphamide analogues in aqueous buffer; release of PDA was observed to a minor extent and only at high (1 M) buffer concentrations. This analogue was essentially inactive in vitro against L1210 and P388 leukemia cells. 6-Phenylcyclophosphamide and its 4-hydroperoxy derivative were potent inhibitors of blood acetylcholinesterase and were lethal at therapeutic doses in mice. In contrast, 4-hydroperoxy-6-(4-pyridyl)cyclophosphamide did not inhibit acetylcholinesterase and showed significant antitumor activity in vitro and in vivo against both wild-type and cyclophosphamide-resistant L1210 leukemia. The 4-hydroperoxy-6-arylcyclophosphamides were generally active in vitro against both wild-type and cyclophosphamide-resistant L1210 and P388 cells, and several analogues showed significant activity in vivo. Surprisingly, there was no correlation between antitumor activity in vitro and the rate of PDA release in aqueous buffer. Several compounds that showed essentially no release of PDA in aqueous buffer over several hours were highly cytotoxic to leukemia cells following a 1-h exposure in vitro. These results show that activated cyclophosphamide analogues substituted at the 6-position are not cross-resistant in these leukemia cell lines, and that a specific intracellular activation mechanism may be catalyzing PDA release in these analogues.

Sugar nucleoside diphosphates have been prepared using an efficient phosphate coupling reaction that employs a highly reactive zwitterionic phosphoramidate intermediate as the phosphorylating species.

A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2‘-deoxyuridine-5‘monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P−N bond cleavage to yield the nucleoside monophosphate quantitatively. In vitro evaluation of 5-fluoro-2‘-deoxyuridine phosphoramidate prodrugs 2a and 3b against L1210 mouse leukemia cells show potent inhibition of cell growth (IC50 0.5−3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compound 2a is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC50 va...

The multifunctional enzyme apurinic endonuclease 1/redox enhancing factor 1 (Ape1/ref-1) maintains genetic fidelity through the repair of apurinic sites and regulates transcription through redox-dependent activation of transcription factors. Ape1 can therefore serve as a therapeutic target in either a DNA repair or transcriptional context. Inhibitors of the redox function can be used as either therapeutics or novel tools for separating the two functions for in vitro study. Presently there exist only a few compounds that have been reported to inhibit Ape1 redox activity; here we describe a series of quinones that exhibit micromolar inhibition of the redox function of Ape1. Benzoquinone and naphthoquinone analogues of the Ape1-inhibitor E3330 were designed and synthesized to explore structural effects on redox function and inhibition of cell growth. Most of the naphthoquinones were low micromolar inhibitors of Ape1 redox activity, and the most potent analogues inhibited tumor cell growth with IC(50) values in the 10-20 microM range.

Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time- dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)- 6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)- alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF- alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony- stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM- CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.

31P NMR spectroscopy was used to study the solvolysis kinetics of a novel series of alkylating monoester phosphoramidates (4a-d) under model physiologic conditions. Halide ion kinetics were used to determine the rate of aziridinium ion formation. The solvolysis rates showed the expected dependence upon substitution at the reactive nitrogen; comparison of 4a with phosphoramide mustard (1a) indicated that replacement of the amino group by alkoxy decreased the solvolysis rate by approximately 10-fold. The rate of conversion of starting compound (4a-d) to solvolysis product was essentially equal to the rate of halide ion release, suggesting that the aziridinium ion is a short-lived intermediate. 1H NMR and 31P NMR kinetics experiments performed in the absence and presence of trapping agent (dimethyldithiocarbamate) confirmed that the aziridinium ion was too short-lived to be observed via NMR. These compounds were also tested for cytotoxicity against L1210 leukemia and B16 melanoma cells in vitro; the monoalkylators 4c and 4d showed no activity, 4a was weakly cytotoxic, and 4b was comparable in activity to phosphoramide mustard.

The DNA base excision-repair pathway is responsible for the repair of DNA damage caused by oxidation/alkylation and protects cells against the effects of endogenous and exogenous agents. Removal of the damaged base creates a baseless (AP) site. AP endonuclease1 (Ape1) acts on this site to continue the BER-pathway repair. Failure to repair baseless sites leads to DNA strand breaks and cytotoxicity. In addition to the repair role of Ape1, it also functions as a major redox-signaling factor to reduce and activate transcription factors such as AP1, p53, HIF-1α, and others that control the expression of genes important for cell survival and cancer promotion and progression. Thus, the Ape1 protein interacts with proteins involved in DNA repair, growth-signaling pathways, and pathways involved in tumor promotion and progression. Although knockdown studies with siRNA have been informative in studying the role of Ape1 in both normal and cancer cells, knocking down Ape1 does not reveal the individual role of the redox or repair functions of Ape1. The identification of small-molecule inhibitors of specific Ape1 functions is critical for mechanistic studies and translational applications. Here we discuss small-molecule inhibition of Ape1 redox and its effect on both cancer and endothelial cells. Antioxid. Redox Signal. 10, 1853–1867.

Most of the drugs currently used in the treatment of cancer are highly cytotoxic and possess relatively low therapeutic selectivity. Given the major role of the kidney in the excretion of these drugs or their metabolites, it is perhaps surprising that clinically significant nephrotoxicity occurs with a limited number of drugs. Adriamycin induces a nephrotic syndrome in rats characterized by severe acute glomerular injury and sustained proteinuria. Tubular lesions subsequently develop, and the formation of tubular casts with resulting interstitial changes is accompanied by the appearance of glomerulosclerosis. Although the mechanism of adriamycin-induced nephrotoxicity is not known, it has been suggested that redox cycling may contribute to this toxicity by enhancing membrane lipid peroxidation, resulting in damage to kidney endoplasmic reticulum and mitochondrial membranes [l]. Mitocycin C is another redox-cycling drug with a cytotoxic mechanism of action that is very different from adriamycin. A hemolytic uremic syndrome of unknown pathogenesis has been reported in some patients treated with mitomycin C; this syndrome has been reproduced in’a unilateral perfusion study in the rat [2]. Nephrotoxicity has been reported for the nitrosoureas in both clinical and laboratory studies. Methyl-CCNU administered as a single, high dose produces acute proximal tubular necrosis in the rat; impaired tubular transport was evident 1 h after drug treatment. Proteinuria and decreased urine-concentrating ability accompanied necrosis of the papillary collecting ducts l-6 days later. Low-dose treatment resulted in a delayed-onset chronic progressive nephropathy involving polyuria, enzymuria, and decreased urine-concentrating ability [3,4]. Several lines of evidence point to the

A gemcitabine (2',2'-difluorodeoxycytidine, dFdC) phosphoramidate prodrug designed for the intracellular delivery of gemcitabine 5'-monophosphate was synthesized. The prodrug was about an order of magnitude less active than gemcitabine against wild-type cells, and the nucleoside transport inhibitor dipyridamole reduced prodrug activity. The prodrug was more active than gemcitabine against two deoxycytidine kinase-deficient cell lines. The results suggest that the prodrug is a potent growth inhibitor that can bypass dCK deficiency at higher drug concentrations.

Certain farnesyl diphosphate (FPP) analogs are potent inhibitors of the potential anticancer drug target protein farnesyltransferase (FTase), but these compounds are not suitable as drug candidates. Thus, phosphoramidate prodrug derivatives of the monophosphate precursors of FPP-based FTase inhibitors have been synthesized. The monophosphates themselves were significantly more potent inhibitors of FTase than the corresponding FPP analogs. The effects of the prodrug 5b (a derivative of 3-allylfarnesyl monophosphate) have been evaluated on prenylation of RhoB and on the cell cycle in a human malignant schwannoma cell line (STS-26T). In combination treatments, 1-3 microM 5b plus 1 microM lovastatin induced a significant inhibition of RhoB prenylation, and a combination of these drugs at 1 microM each also resulted in significant cell cycle arrest in G1. Indeed, combinations as low as 50 nM lovastatin + 1 microM 5c or 250 nM lovastatin + 50 nM 5c were highly cytostatic in STS-26T cell culture.

N-2,3-dihydroxypropyl-N-4-chlorobutyl phosphoramidate prodrugs of thymidine and 5-fluoro-2'-deoxyuridine (5-FdU) 2-4 were synthesized as analogues of the known prodrugs 1a,b to assess the effects of the dihydroxypropyl moiety on prodrug activation, log P, and growth inhibitory activity in vitro. The thymidine analogues 2 and 3 were prepared as model compounds for kinetics and log P studies. 31P NMR kinetics following hydrogenolysis of 2 showed that the thymidine N-dihydroxypropyl phosphoramidate released thymidine monophosphate with a half-life of 212 min under model physiologic conditions compared to approximately 2 min for the corresponding N-methyl phosphoramidate reported previously. The measured log P for compound 3 was 1.1 log units lower than that of the analogous 1b, confirming that the dihydroxypropyl group significantly decreased prodrug lipophilicity. The dihydroxypropyl prodrug 4 showed cell growth inhibition activity in the NCI 60 cell line panel similar to that of the N-methyl analogue 1a previously reported.

Recently, we reported a novel approach for the intracellular delivery of the anti-cancer nucleotide 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) using phosphoramidate-based prodrugs. These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation, liberating phosphoramidate anion, which in turn undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quantitatively. This approach has now been extended to cytarabine [1-beta-D-arabinofuranosylcytosine (Ara-C)], an anti-cancer nucleoside that is limited in its utility because of poor intracellular transport characteristics and weak activity as a substrate for tumor cell kinases. The cytarabine phosphoramidate prodrug 1 has been synthesized and evaluated in comparison with cytarabine for growth inhibitory activity against wild-type, nucleoside transport-deficient, and nucleoside kinase-deficient CEM leukemia cell lines. The prodrug was comparable in growth inhibitory activity (IC50 = 32 nM) to cytarabine (IC50 = 16 nM) in wild-type CCRF-CEM cells following drug treatment for 72 h. The nucleoside transport-deficient CEM/AraC8C exhibited a high level of resistance (6400-fold) to cytarabine but was more sensitive (210-fold resistant vs CCRF-CEM cells) to prodrug 1. Similarly, the deoxycytidine kinase-deficient cell line (CEM/dCK-) was highly resistant to cytarabine (13900-fold) but more sensitive (106-fold resistant vs CCRF-CEM cells) to prodrug 1. These results indicate that prodrug 1 is significantly more potent than cytarabine against transport- and kinase-deficient cell lines and are consistent with a mechanism involving intracellular delivery of cytarabine 5'-monophosphate.

[reaction: see text] A novel method for the preparation of nucleoside triphosphates has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes efficient coupling with tris(tetra-n-butylammonium) hydrogen pyrophosphate to generate nucleoside triphosphate.

Reductive Amination with Sodium Cyanoborohydride: N,N-Dimethylcyclohexylamine product: N,N-dimethylcyclohexylamine byproduct: cyclohexanol product: Cyclohexylamine product: N-Methylcyclohexylamine product: α-Phenylethylamine product: N-Methylphenethylamine product: N-Isobutylaniline product: N-Methylpiperidine Keywords: condensation, miscellaneous; reduction, miscellaneous; sodium cyanoborohydride

[reaction: see text] A novel method for the immobilization of nucleotides has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes nucleophilic attack by long-chain alkylamine-controlled pore glass (LCAA-CPG) to generate an immobilized nucleotide. Quantification of nucleotide loading was accomplished by acidic hydrolysis of the P-N bond and subsequent HPLC analysis of TMP in the presence of an internal standard. Typical nucleotide loadings of 51-59 micromol/g of support were observed.

A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2'-deoxyuridine-5'monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quantitatively. In vitro evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs 2a and 3b against L1210 mouse leukemia cells show potent inhibition of cell growth (IC(50) 0.5-3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compound 2a is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC(50) value for thymidylate synthase inhibition that is comparable to FUdR in thymidine kinase deficient cells. Further studies revealed that 3b rapidly decomposes to the nucleotide in cell culture medium, suggesting that the naphthoquinone analogue is not sufficiently stable to function as a nucleotide prodrug.

A novel nitroheterocyclic bis(haloethyl)phosphoramidate prodrug linked through lysine to a pteroic acid has been prepared and evaluated as a potential alkylating agent to target tumor cells that overexpress the folate receptor. The prodrug exhibited IC(50) values in the micromolar range and was 10-400-fold less cytotoxic in vitro than the phosphoramidate that lacks the lysine-pteroyl moiety. The data does not support a contribution of the folate receptor to cytotoxicity. In an attempt to determine the basis for the decreased cytotoxicity in the pteroyl-lysyl analogue, compounds were prepared in which the lysine-pteroyl moiety was replaced with lysine alone or with an n-propyl group. The n-propyl and the lysyl analogues were on average 3.8- and 21-fold less potent than the unsubstituted bis(haloethyl)phosphoramidate, respectively. Chemical reduction of the prodrugs followed by (31)P NMR kinetics demonstrated that all of the phosphoramidate anions cyclized to the aziridinium ion at similar rates and gave comparable product distributions, suggesting that changes in chemical activation did not account for the differences in cytotoxicity. It is likely that folate receptor-mediated transport is not sufficient to deliver adequate intracellular concentrations of the cytotoxic phosphoramide mustard.

A series of novel nitroheterocyclic phosphoramidates has been evaluated for antitumor activity in murine and xenograft tumor models and for toxicity in mice. Significant increases in lifespan and long-term survivors were noted in L1210 leukemia and B16 melanoma models, and both complete and partial tumor regressions were observed in the MX-1 breast cancer xenograft model. All compounds exhibited some degree of toxicity to granulocyte/macrophage progenitors in the bone marrow of mice. Two drugs were selected for further toxicologic, histopathologic, and pharmacokinetic evaluations. Toxicity of potential clinical significance was observed only in the bone marrow at the highest drug dose; otherwise no significant abnormalities in blood chemistries or organ histopathology were noted. The bone marrow lesions consisted of reduced numbers of progenitor cells in the myeloid and erythroid series; platelets were not affected. The compounds were eliminated rapidly by first-order kinetics, with half-lives in the 4-12 min range. The best of these compounds exhibits excellent antitumor activity and minimal toxicity at therapeutically effective doses in mice.

A series of thymidine and tetrahydrofurfuryl phosphoramidates bearing haloethyl or piperidyl substituents was synthesized and used to investigate the activation mechanisms of nucleoside phosphoramidate prodrugs. Structure assignments for the tetrahydrofurfuryl reaction products were confirmed by comparison to authentic samples. Structural assignments for thymidine phosphoramidate reaction products were made by analogy to the tetrahydrofurfuryl products. Generation of the phosphoramidate anion leads to cyclization and subsequent nucleophilic attack at carbon and phosphorus of the resulting aziridinium ion intermediate to give the observed products. Nucleophilic attack by water at carbon and phosphorus occurs without selectivity, supporting a mechanism of action of haloethylamine nucleoside prodrugs involving intracellular release of the nucleotide. Activation of the benzotriazolyl piperidyl phosphoramidates is followed by P-N bond hydrolysis; this reaction is subject to specific acid catalysis and to nucleophilic catalysis by 1-hydroxybenzotriazole. These results suggest that the mechanism of action of the piperidyl nucleoside phosphoramidates involves the intracellular release of the active nucleotide following P-N bond cleavage, presumably by the action of an endogenous phosphoramidase.

A series of novel haloethyl and piperidyl phosphoramidate FdUMP prodrug analogues has been synthesized, and the growth inhibitory activity of these compounds has been evaluated against L1210 mouse leukemia cells. All compounds exhibited potent inhibition of L1210 cell proliferation with IC(50) values in the nanomolar range. Growth inhibition was reversed by the addition of 5 microM thymidine, suggesting a mechanism of action involving the intracellular release of FdUMP. (31)P NMR studies carried out on model haloethyl phosphoramidates confirm the release of nucleotide via cyclization of the phosphoramidate anion to the aziridinium ion intermediate followed by hydrolysis of the P-N bond. The data suggests that50% of the prodrug is converted to FdUMP intracellularly by this pathway. Piperidyl phosphoramidate analogues are also converted to nucleotide intracellularly, presumably by the action of an endogenous phosphoramidase.

Background. The uncontrolled growth that characterizes cancer cells is attributed to aberrations in pathways that regulate progress through the cell cycle. Mitotic centromere-associated kinesin (MCAK) is a microtubule-depolymerizing kinesin that functions in spindle assembly and is critical for regulating kinetochore microtubule (MT) dynamics to control proper attachment of chromosomes to the spindle. The critical role that MCAK plays in mitosis makes it an attractive candidate for further investigation. Several studies suggest that MCAK levels correlate with poor prognosis in certain cancers. Knockdown of MCAK in cells derived from multiple cancers reduced cell proliferation. Thus, inhibitors of MCAK have considerable potential as a novel cancer therapeutic. We have synthesized a phosphotyrosine mimetic prodrug, RH6-22, that is cytostatic at low micromolar concentrations for leukemia cells and delivers a non-hydrolyzable phosphotyrosine peptidomimetic intracellularly. A screen for candidate target proteins using immobilized RH6-22 phosphonate identified MCAK as a potential target for this peptidomimetic. RH6-22 induces polyploidy in DG75 cells with no measureable cell death. Disruption of the microtubule network was seen in WEHI 231 cells treated with RH6-22 and stained with antibodies against α-tubulin. These experiments suggested defects in both mitosis and cytokinesis consistent with MCAK inhibition. We hypothesized that RH6-22 was interacting with MCAK at the ATP-biding site. Methods. A competitive ATP binding assay was run to confirm the interaction of RH6-22 at the ATP binding site. A focused library of RH6-22 analogs was synthesized and analyzed for growth inhibitory activity in DG75 cells. Molecular modeling studies were carried out using the crystal structure of mammalian MCAK (pdb 1V8J) with the goal of developing higher affinity small molecule inhibitors. The highest affinity compounds identified in silico were chosen for synthesis and evaluation in a growth inhibition assay in DG75 cells. Results. Concentrations of ATP as low as 6μM can selectively elute MCAK from the immobilized RH6-22 ligand, indicating that the ligand binds at the ATP binding site. The small library of RH6-22 analogs tested in growth inhibition assays resulted in compounds with IC50 values from 4–50μM. Docking experiments in silico exploring variations to the non-phosphate portion of RH6-22 provided compounds with significant improvements in predicted binding affinity. The best compounds chosen for synthesis and biological evaluation are similar in that they contain a dihydroxybenzofuran moiety that hydrogen bonds with Arg78 and allows a favorable pi-pi stacking interaction with His170. Synthesis of the dihydroxybenzofuran is accomplished through an enzyme-catalyzed oxidation of catechol followed by the Michael addition of methyl 2-chloroacetoacetate and subsequent cyclization. These new compounds exhibited sub-micromolar growth inhibitory activity. Conclusion. We have used our phosphopeptidomimetic prodrug RH6-22 as a lead compound and structure-based drug design to synthesize novel inhibitors targeted to MCAK. The best compounds demonstrate submicromolar growth inhibitory activity in cell-based assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B184.

Background. Many protein-protein interactions in cells are mediated by domains that bind to motifs that contain phosphorylated serine and threonine residues. To create small molecules that inhibit these interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. As a proof of principle, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. 14-3-3 is a highly conserved and ubiquitously expressed family of proteins that is involved in many vital cellular processes including signal transduction, apoptosis, cell-cycle regulation, growth, and development. 14-3-3 proteins bind to many targets including the tumor suppressor p53, Raf family kinases, FOXO transcription factors, the pro-apoptotic protein BAD, and Cdc25 phosphatases which makes the 14-3-3 family an interesting target for cancer therapeutics. Methods. The small molecule prodrug KK7–83 and the corresponding free phosphonate ligand KK8–81 were synthesized. The growth inhibitory activity of both compounds was measured in DG75 leukemia cells. The potential cytotoxic effects of the prodrug were investigated by monitoring the cleavage of PARP, a well known caspase substrate. To explore a direct involvement of 14-3-3 proteins in the actions of KK7–83, we developed an assay using a luciferase readout to monitor the 14-3-3-dependent inhibition of FOXO transcription factors. Results. In growth inhibition studies, prodrug KK7–83 was a potent inhibitor with an IC50 = 5 M, while the free phosphonate KK8–81 showed no meaningful growth inhibition up to 100 M. Significant PARP cleavage was observed by Western blot following prodrug treatment at 12 M, suggesting that KK7–83 induces apoptosis at low micromolar concentrations. In the luciferase assay, the ectopic expression of FOXO3a led to a substantial increase in FOXO3a-mediated gene transcription compared to control, which was inhibited upon co-transfection of Akt1 as expected. FOXO3a-stimulated transcription was recovered in a dose dependent manner by treatment of cells with increasing concentrations of KK7–83, consistent with the prodrug's inhibition of 14-3-3-mediated retention of FOXO3a in the cytoplasm. The concentration of KK7–83 required to recover 50% of the FOXO3a induced gene expression was consistent with the IC50 value for growth inhibition, further suggesting that the cytotoxicity of KK7–83 is a function of its ability to bind 14-3-3. Conclusions. KK7–83 is a potent inhibitor of cancer cell growth leading to apoptosis at low micromolar concentrations through direct interactions with the 14-3-3 family of proteins. Development of this small molecule validates our prodrug strategy and the further exploration of the 14-3-3 family for the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A124.

Sodium diethyldithiocarbamate (DDTC) has been investigated as a biochemical modulator of the toxicity associated with clinically used cancer chemotherapeutic agents. In the present study, we assessed the ability of DDTC to accelerate recovery of the granulocyte/macrophage progenitor cel (GM-CFC) population following treatment with the myelosuppressive drugs carboplatin (CBDCA), tetrachloro(d,l-trans)1,2-diaminocyclohexane platinum(IV) (tetraplatin), 5-fluorouracil (5-FU), and etoposide (VP-16) in B6D2F1 mice. Myelotoxicity was assessed 24 h after the injection of the anticancer drug using a GM-CFC clonogenic assay. In the case of all four anticancer drugs, the timing of DDTC administration appeared to be a critical parameter with regard to protection. A delay time of 1 h between the injection of the myelotoxic drug and treatment with DDTC (30 mg/kg) resulted in a significant reduction in cytotoxicity to GM-CFC, whereas a longer delay time did not. These results suggest that the timing of DDTC administration may be essential in modulating the myelosuppression associated with many chemotherapeutic regimens used in the clinic.

A series of perhydrooxazine analogues of aldophosphamide has been prepared, and their 31P NMR kinetics and in vitro cytotoxicity have been evaluated. These compounds were developed on the basis of the idea that ring opening and tautomerization to an enamine intermediate might provide a mechanistic alternative to the beta-elimination reaction for release of phosphoramide mustard. The 4,4,6-trimethyltetrahydro-1,3-oxazine moiety was selected on the basis of its rapid rate of iminium ion generation and relatively slow rate of hydrolysis. These analogues underwent phosphorodiamidate release by three distinct mechanisms: hydrolysis to aldophosphamide and subsequent beta-elimination; cyclization to produce the 4-hydroxycyclophosphamides, which release phosphorodiamidate by ring opening and elimination; and tautomerization to the enamine with rapid expulsion of phosphorodiamidate. Kinetic studies demonstrated that hydrolysis to the aldehyde contributed minimally to the overall activation process and that the enamine pathway represented the major route of activation. For those analogues that could undergo cyclization, this pathway competed effectively with enamine release, and these analogues were essentially equivalent to their 4-hydroxycyclophosphamide counterparts in cytotoxicity. A series of tetra-N-substituted phosphorodiamidates that cannot undergo cyclization was prepared to explore the effects of cyclization on the cytotoxicity of these analogues. The tetrakis(chloroethyl)phosphorodiamidates were highly potent in vitro against both cyclophosphamide-sensitive and -resistant L1210 and P388 cell lines, and one of these analogues had significant antitumor activity against L1210 leukemia in vivo. These results demonstrate that the enamine mechanism provides a viable pathway for delivery of phosphorodiamidates and that this approach can be used to deliver phosphorodiamidates that are non-cross-resistant in cyclophosphamide-resistant cell lines.

A series of 5- and 6-substituted cyclophosphamide analogues has been prepared, and their 31P NMR kinetics of phosphoramide mustard (PDA) release and in vitro and in vivo cytotoxicity have been evaluated. cis-4-Hydroxy-5-methoxycyclophosphamide equilibrated very slowly and to a minor extent with the ring-opened aldophosphamide analogues in aqueous buffer; release of PDA was observed to a minor extent and only at high (1 M) buffer concentrations. This analogue was essentially inactive in vitro against L1210 and P388 leukemia cells. 6-Phenylcyclophosphamide and its 4-hydroperoxy derivative were potent inhibitors of blood acetylcholinesterase and were lethal at therapeutic doses in mice. In contrast, 4-hydroperoxy-6-(4-pyridyl)cyclophosphamide did not inhibit acetylcholinesterase and showed significant antitumor activity in vitro and in vivo against both wild-type and cyclophosphamide-resistant L1210 leukemia. The 4-hydroperoxy-6-arylcyclophosphamides were generally active in vitro against both wild-type and cyclophosphamide-resistant L1210 and P388 cells, and several analogues showed significant activity in vivo. Surprisingly, there was no correlation between antitumor activity in vitro and the rate of PDA release in aqueous buffer. Several compounds that showed essentially no release of PDA in aqueous buffer over several hours were highly cytotoxic to leukemia cells following a 1-h exposure in vitro. These results show that activated cyclophosphamide analogues substituted at the 6-position are not cross-resistant in these leukemia cell lines, and that a specific intracellular activation mechanism may be catalyzing PDA release in these analogues.

Quiescent LLC-PK1 cells have been shown to be a good model of cis-diamminedichloroplatinum(II) (DDP)-induced nephrotoxicity. In these nonproliferating porcine kidney epithelial cells, DDP inhibition of protein synthesis rate is the major correlate of cytotoxicity. We report here the use of this cell line to investigate the role of endogenous sulfur-containing nucleophiles in DDP-induced nephrotoxicity. Reaction of DDP with glutathione (GSH), cysteine, or methionine for up to 24 hr led to concentration- and time-dependent loss of its toxic effects, whereas dissolution of DDP alone did not alter its reduction of viability or protein synthesis rate in LLC-PK1 cells. Treatment of these cells with differing cytotoxic concentration of DDP produced an identical transient increase in intracellular GSH, whereas compounds known to bind GSH, trans-diamminedichloroplatinum(II) (t-DDP) and diethyl maleate (DEM), rapidly depleted LLC-PK1 intracellular GSH levels. Buthionine sulfoximine (BSO) treatment decreased intracellular GSH to 10% of control without altering cell viability or protein synthesis rate. However, BSO-pretreated LLC-PK1 cells exhibited enhanced DDP-induced toxicity. CdCl2 treatment produced a 30-fold induction of metallothionein-like, cadmium-binding proteins and a 10-fold increase in metallothionein isoform I (MT-I) mRNA, but this induction had no effect on DDP-induced reduction of viability or protein synthesis rate. Protracted DDP exposure did not induce MT-I mRNA levels in LLC-PK1 cells.