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Objective: To analyze the correlation between postoperative complications and combined deflection angle classification adduction type (CDAC-ADT) of femoral neck fractures after cannulated screw internal fixation., Methods: The clinical data of 121 patients with CDAC-ADT femoral neck fracture admitted between January 2018 and December 2021 and met the selected criteria were retrospectively analyzed. There were 69 males and 52 females, the age ranged from 19 to 79 years (mean, 48.1 years). The causes of injury included 52 cases of traffic accident, 24 cases of falling from height, and 45 cases of fall. The time from injury to operation ranged from 2 to 12 days, with an average of 6.0 days. Among them, there were 18 cases of CDAC-ADT type Ⅰ, 46 cases of type Ⅱ, and 57 cases of type Ⅲ; 6 cases of Garden type Ⅱ, 103 cases of type Ⅲ, and 12 cases of type Ⅳ; and according to the location of the fracture line, there were 26 cases of subcapitate type, 88 cases of transcervical type, and 7 cases of basal type. All patients were treated with cannulated screw internal fixation. The occurrence of complications (including internal fixation failure, fracture nonunion, and osteonecrosis of the femoral head) was recorded, and the correlation between complications and CDAC-ADT typing, Garden typing, and fracture line location were analyzed., Results: The patients were followed up 8-44 months, with a mean of 24.9 months. There were 10 cases of internal fixation failure, 7 cases of fracture nonunion, and 30 cases of osteonecrosis of the femoral head after operation. Correlation analysis showed that patients' CDAC-ADT typing was significantly correlated with the overall incidence of complication and the incidence of internal fixation failure, fracture nonunion, and osteonecrosis of the femoral head ( P <0.05), and the Pearson coefficient of contingency were 0.435, 0.251, 0.254, and 0.241, respectively. Garden typing did not correlate with the overall incidence of complication and the incidence of internal fixation failure and fracture nonunion ( P >0.05), but correlated with the incidence of osteonecrosis of the femoral head ( P <0.05), and the Pearson coefficient of contingency was 0.251. Fracture line position typing had no correlation with the overall incidence of complication and the incidence of internal fixation failure, fracture nonunion, and osteonecrosis of the femoral head ( P >0.05)., Conclusion: CDAC-ADT typing has obvious correlation with postoperative complications of femoral neck fracture and can be used to predict complications of femoral neck fracture.

Carbon-based superoxide dismutase (SOD) mimetic nanozymes have recently been employed as promising antioxidant nanotherapeutics due to their distinct properties. The structural features responsible for the efficacy of these nanomaterials as antioxidants are, however, poorly understood. Here, the process-structure-property-performance properties of coconut-derived oxidized activated charcoal (cOAC) nano-SOD mimetics are studied by analyzing how modifications to the nanomaterial's synthesis impact the size, as well as the elemental and electrochemical properties of the particles. These properties are then correlated to the in vitro antioxidant bioactivity of poly(ethylene glycol)-functionalized cOACs (PEG-cOAC). Chemical oxidative treatment methods that afford smaller, more homogeneous cOAC nanoparticles with higher levels of quinone functionalization show enhanced protection against oxidative damage in bEnd.3 murine endothelioma cells. In an in vivo rat model of mild traumatic brain injury (mTBI) and oxidative vascular injury, PEG-cOACs restore cerebral perfusion rapidly to the same extent as the former nanotube-derived PEG-hydrophilic carbon clusters (PEG-HCCs) with a single intravenous injection. These findings provide a deeper understanding of how carbon nanozyme syntheses can be tailored for improved antioxidant bioactivity, and set the stage for translation of medical applications., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

A recently optimized rapid, cheap, and accurate coulometric method has been exploited to determine the antioxidant capacity of bergamot (Citrus bergamia Risso) by-products, including first (FPJ) and second press juices (SPJ), in comparison to analogous products from several citrus species. Extracts from the entire edible part (i.e., juice and pulp) and de-oiled peel of bergamot were also assayed. The Coulometrically Determined Antioxidant Capacity (CDAC) data, expressed as moles of electrons per mass of sample, were evaluated with other parameters such as total phenolic compounds, ascorbic acid, total carotenoids, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition. The CDAC of bergamot FPJ (39 mmol e - kg -1 ) was comparable with other citrus juices (20-65 mmol e - kg -1 range), whereas the CDAC of bergamot SPJ (816 mmol e - kg -1 ) was strikingly higher than the counterparts from other citrus fruits. This value approached that of bergamot peel extracts (822 mmol e - kg -1 ). Bergamot peel and SPJ also exhibited the highest DPPH inhibition. The CDAC values were associated with the HPLC-determined content of flavonoids, namely neoeriocitrin, naringin, and neohesperidin, which were 4-10-fold more concentrated in bergamot SPJ and peel than in SPJ from other citrus species. These findings contribute to point at bergamot by-products as rich sources of antioxidant compounds on a quantitative basis, highlighting their enormous potential for pharmaceutical, nutraceutical and food applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive pediatric malignancy primarily affecting infants and young children. Intensive multimodal therapies currently given to MRT patients are not sufficiently potent to control this highly malignant tumor. Therefore, additive or alternative therapy for these patients with a poor prognosis is necessary. We herein demonstrated that the inhibition of runt-related transcription factor 1 (RUNX1) by novel alkylating conjugated pyrrole-imidazole (PI) polyamides, which specifically recognize and bind to RUNX-binding DNA sequences, was highly effective in the treatment of rhabdoid tumor cell lines in vitro as well as in an in vivo mouse model. Therefore, suppression of RUNX1 activity may be a novel strategy for MRT therapy., (© 2020 Wiley Periodicals LLC.)

Here we report the design and synthesis of a chlorambucil-alkynyl (CHL-CCH) ligand, mononuclear gold(i) complex K[(CHL-CC)AuCl], 1, and heteronuclear complex (CHL-CC)Au(μ2-η2-CS3)Ti(η5-Cp)2, 2 for renal cancer. Complex 2 is significantly more cytotoxic than complex 1 and cisplatin against renal cancer cells with a high selectivity index value. The mechanism of action of these complexes against renal cancer cells was studied in detail by experimental and computational methods.

The runt-related transcription factor (RUNX) family has been associated with cancer development. The binding of RUNX family members to specific DNA sequences is hypothesized to promote the expression of downstream genes and cause cancer proliferation. On the basis of this proposed mechanism of cancer growth, we developed conjugate 1, which inhibits the binding of RUNX to its target DNA. Conjugate 1 is a DNA-alkylating pyrrole-imidazole (PI) polyamide conjugate containing chlorambucil as an anticancer agent. Conjugate 1 was reported to have a marked anticancer effect in mouse models of acute myeloid leukemia. Although the effectiveness of 1 has been demonstrated in vivo, the detailed mechanism by which it alkylates DNA is unknown. Here, we chemically elucidated the molecular characteristics of conjugate 1 to confirm its potential as a RUNX-inhibiting drug. We also generated an alternative conjugate 2, which targets the same DNA sequence, by replacing one pyrrole with β-alanine. Comparison of the characteristics of conjugates 1 and 2 suggested that reaction selectivity and binding affinity to the RUNX-binding sequence were improved by the introduction of β-alanine. These findings indicate the possibility of DNA-alkylating PI polyamides as candidates for cancer chemotherapeutics.

A FRET donor-acceptor xanthene-coumarin conjugate has been designed for redox-regulated synergic treatment of photodynamic therapy and chemotherapy with real-time monitoring. The "locked" FRET pair was selectively "unlocked" by biological reducing thiols via rupture of a sacrificial disulfide linker. A distinct change in fluorescence color and selective cancer cell toxicity were observed in vitro.

The NF-κB pathway is overexpressed in non-small cell lung cancers (NSCLC) and contributes to the poor prognosis and high mortality characterizing this malignancy. Silencing the p50 and p65 NF-κB subunits in the NSCLC H1299 cell line led to profound loss in cell viability and downregulated anti-apoptotic proteins survivin and Mcl1. We also showed that a survivin suppressant, the dioxonaphthoimidazolium YM155, and its structural analog AB1 arrested the growth of H1299 cells at nanomolar concentrations. Both compounds were apoptogenic and suppressed survivin and other anti-apoptotic proteins (Mcl1, Bcl-2, Bcl-xl) in a dose- and/or time-dependent manner. YM155 and AB1 did not affect the expression of key proteins (IκBα, p65, p50) involved in NF-κB signaling. Stable IκBα levels suggest that the NF-κB/IκB complex and proteins upstream of IκBα, were not targeted. Neither did the compounds intercept the nuclear translocation of the p50 and p65 subunits. On the other hand, YM155 and AB1 suppressed the phosphorylation of the p50 subunit at Ser337 which is critical in promoting the binding of NF-κB dimers to DNA. Both compounds duly impeded the binding of NF-κB dimers to DNA and attenuated transcriptional activity of luciferase-transfected HEK293 cells controlled by NF-κB response elements. We propose that the "silencing" the NF-κB pathway effected by these compounds contributed to their potent apoptogenic effects on H1299. Notwithstanding, the mechanism(s) involved in their ability to abolish phosphorylation of p50 remains to be elucidated. Taken together, these results disclose a novel facet of functionalized dioxonaphthoimidazoliums that could account for their potent cell killing property.

The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

The blood brain barrier (BBB) represents the biggest challenge for therapeutic drugs to enter the brain. In our study, we selected chlorambucil (CHL), an alkylating agent, as the model therapeutic agent, and used scopine as a novel brain-targeting moiety. Here, we synthesized Chlorambucil-Scopine (CHLS) prodrug and evaluated its brain-targeting efficacy. The tissue distribution study after i.v. injection revealed that the AUC0-t and Cmax of CHLS in the brain were 14.25- and 12.20-fold of CHL, respectively. Specifically, CHLS accumulated in bEnd.3 and C6 cells in an energy-dependent manner. In C6 cells, superior anti-glioma activity with a significantly decreased IC50 of 65.42 nM/mL was observed for CHLS compared to CHL (IC50 > 400 nM/mL). The safety evaluation, including acute toxicity, pathology, and hematology study, showed minimal toxicity toward nontargeting tissues, and also reached a lower systemic toxicity at 5 mg/kg (i.v.). Our results suggested that scopine is a potential brain-targeting moiety for enhancing the brain uptake efficiency of CHL.

Cancer is one of the most fatal diseases in the world and it has been years that finding new drugs and chemotherapeutic techniques with lowest side effects become one of the most important challenging matters needs really hard efforts. Chlorambucil (CBL), an ancient direct-acting alkylating anticancer agent, is commonly used for initial treatment of some kinds of cancers but the use of CBL is often limited because of the unpleasant side effects due to its lack of specificity for targeting cancer cells. In this research we tried to increase the specificity of CBL by producing a novel conjugate by using glutamine amino acid (Glut). Based on previous studies, poly amines and nitrogen compounds noticeably are used by cancer cells increasingly; therefore we decided to increase the efficiency and specificity of CBL by designing and producing a novel anti cancer conjugate using glutamine amino acid as an uptake enhancer, CBL, and Adipic acid Dihydrazide (ADH) as a spacer and linker. The biological tests were carried out on HT29 colorectal cancer cell line to evaluate its anticancer properties. Biological tests like MTT assay, finding IC50, evaluating the induced mechanism of the death of our novel CBL-Glutamine conjugate on HT29 cells, testing abnormal toxicity of this conjugate on mice in comparison with CBL drug were careid out. We found that not only CBL-Glutamine conjugate preserved its anti cancer property with regard to CBL drug, but also it represent lower abnormal toxicity in mice. Apoptosis was detected as its mechanism of the death. Our present study provides a promising strategy for targeting cancer cells using amino acids nano-conjugate drugs. The future perspectives have also been highlighted in continuing similar and relative researches.

Amino acids were transformed and coupled to chlorambucil, a well-known chemotherapeutic agent, in an attempt to create new anticancer drugs with selectivity for breast cancer cells. Among the amino acids available, tyrosine was selected to act as an estrogenic ligand. It is hypothesized that tyrosine, which shows some structural similitude with estradiol, could possibly mimic the natural hormone and, subsequently, bind to the estrogen receptor. In this exploratory study, several tyrosine-drug conjugates have been designed. Thus, ortho-, meta- and para-tyrosine-chlorambucil analogs were synthesized in order to generate new anticancer drugs with structural diversity, more specifically in regards to the phenol group location. These new analogs were produced in good yield following efficient synthetic methodology. All the tyrosine-chlorambucil hybrids were more effective than the parent drug, chlorambucil. In vitro biological evaluation on estrogen receptor positive and estrogen receptor negative (ER(+) and ER(-)) breast cancer cell lines revealed an enhanced cytotoxic activity for compounds with the phenol function located at position meta. Molecular docking calculations were performed for the pure L-ortho, L-meta- and L-para-tyrosine phenolic regioisomers. The synthesis of all tyrosine-chlorambucil hybrid regioisomers and their biological activity are reported herein. Possible orientations within the targeted protein [estrogen receptor alpha (ERα)] are discussed in relation to the biological activity.

The amino analogues of pentamidine with a polymethylene (n = 3 - 6) chain and their chlorambucil derivatives were synthesized. The obtained compounds revealed cytotoxic effect on MCF-7 human breast cancer cell line (IC50 = 22 - 95 +/- 2 pM), mainly by the induction of apoptosis. The topoisomerase I/II inhibition assay and the ethidium displacement assay with the use of pBR322 plasmid DNA were used to the study of mechanism by which the obtained compounds could act. All the compounds are able to bind with DNA and interfere in vitro with the activity of topoisomerase (I and II). The determination of association constants with the use of calf thymus DNA, T4 coliphage DNA, poly(dA-dT)2 and poly(dG-dC)2 showed that the tested compounds bind within minor groove of B-DNA, but not selectively. The alkylating activity of chlorambucil derivatives determined in vitro using a Preussmann test was similar to the activity of chlorambucil. The influence of all the compounds on the amidolytic activity of plasmin and trypsin was also examined. The plasmin activity was inhibited by pentamidine, chlorambucil and aromatic bis-amines (IC50 = 0.1 - 8 mM), whereas the trypsin activity was influenced only by pentamidine.

Evaluation of the cytotoxicity of a novel G3 PAMAM-NH(2) dendrimer-chlorambucil conjugate employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that the conjugate was more potent antiproliferative agent than chlorambucil. It was found that dendrimer-chlorambucil conjugate was more active inhibitor of collagen biosynthesis than chlorambucil. Our experiments carried out with flow cytometry assessment of annexin V binding and fluorescent microscopy assay revealed that PAMAM-CH conjugate inhibited the proliferation of MCF-7 and MDA-231 malignant cells by increasing the number of apoptotic and necrotic cells. The apoptotic effect of PAMAM-CH conjugate was found to be stronger than that caused by chlorambucil., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A series of D- and L-tyrosine-chlorambucil analogs was synthesized as anticancer drugs for chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of D- and L-tyrosine. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent (ER+ and ER-) breast cancer cell lines. The novel analogs showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals both, the influence of the length of the spacer chain and the stereochemistry of the tyrosine moiety. Interestingly, the D- and L-tyrosinol-chlorambucil derivatives with 10 carbon atoms spacer are selective towards MCF-7 (ER+) breast cancer cell line., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

To systematically assess the impact of glycosylation and the corresponding chemoselective linker upon the anticancer activity/selectivity of the drug chlorambucil, herein we report the synthesis and anticancer activities of a 63-member library of chlorambucil-based neoglycosides. A comparison of N-alkoxyamine-, N-acylhydrazine-, and N-hydroxyamine-based chemoselective glycosylation of chlorambucil revealed sugar- and linker-dependent partitioning among open- and closed-ring neoglycosides and corresponding sugar-dependent variant biological activity. Cumulatively, this study represents the first neoglycorandomization of a synthetic drug and expands our understanding of the impact of sugar structure upon product distribution/equilibria in the context of N-alkoxyamino-, N-hydroxyamino-, and N-acylhydrazine-based chemoselective glycosylation. This study also revealed several analogues with increased in vitro anticancer activity, most notably D-threoside 60 (NSC 748747), which displayed much broader tumor specificity and notably increased potency over the parent drug.

p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE), a nitrogen mustard analogue and chlorambucil's active metabolite used as chemotherapeutic agent, has been shown that, in addition to its clastogenic activity, induces chromosome delay. In the present study an efford has been made (a) to investigate if the steroidal analogues of PHE (EA-92, EA-97, AK-333, AK-409 and AK-433) exert the same genetic activity as the parent compound, (b) to further analyze the aneugenic activity of nitrogen mustard analogues, (c) to investigate the mechanism by which they exert aneugenic potential and (d) to correlate the genetic activity with chemical structure. For this purpose the Cytokinesis Block Micronucleus (CBMN) assay was conducted in human lymphocytes in vitro and the micronucleus (MN) frequency was determined to investigate their genetic activity. The mechanism of micronucleation was determined in combination with Fluorescence In Situ Hybridization (FISH) using pancentromeric DNA probe. Since one of the mechanisms that chemicals cause aneuploidy is through alterations in the mitotic spindle, we also investigated the effect of the above compounds on the integrity and morphology of the mitotic spindle using double immunofluorescence of beta- and gamma-tubulin in C(2)C(12) mouse cell line. We found that PHE and its steroidal analogues, EA-92, EA-97, AK-333, AK-409 and AK-433, affect cell proliferation in human lymphocytes and C(2)C(12) mouse cells. All studied compounds are capable of inducing chromosome breakage events, as indicated by the enhanced C(-)MN frequencies. The less lipophilic compounds are the most genetically active molecules. PHE and only two of the studied analogues, AK-409 and AK-433, the most hydrophilic ones, showed aneugenic potential, by increasing the frequencies of MN containing a whole chromosome. The aneugenic potential of the above referred analogues is associated with amplification of centrosome number, since they caused high multipolar metaphase frequencies., (Copyright 2010 Elsevier B.V. All rights reserved.)

Chlorambucil belongs to a group of nitrogen mustards which are used for the treatment of variety of cancers. Hence, a chlorambucil derivative has been radiolabeled with [(99m)Tc(CO)(3)(H(2)O)(3)](+) core and its efficacy as a tumor targeting agent has been evaluated. Radiochemical yield of the complex was >98% as observed by HPLC. The in vitro studies in MCF-7 breast cancer cells showed about 30% inhibition of the radiolabeled complex in presence of the cold chlorambucil derivative. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed an uptake of 3.2+/-0.3%ID/g at 3h.p.i.

Four ester prodrugs derived from the bifunctional alkylating agent chlorambucil, and one of its nitro-derivatives, 3-nitrochlorambucil conjugated to prasterone and pregnenolone, were synthesized and tested for their cytotoxic activity against eight human cell lines, using the standard MTT assay. A comparison between the esters and the controls, namely chlorambucil and 3-nitrochlorambucil would suggest that all four esters possess to varying degrees, specificity towards the breast adenocarcinoma cell line (MDA-mb468) than the other seven cells' lines tested. The overall findings are encouraging since it infers that these lipophilic esters not only have the ability to traverse specific cell membranes but also exhibit cytotoxicity towards most of the cell lines tested.

The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar liposomes (86-125 nm) and are hydrolyzed by phospholipase A(2), resulting in chlorambucil release. Liposomal formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in the presence of phospholipase A(2), with IC(50) values in the 8-36 microM range.

Alkylating agents are a major class of anticancer drugs for the treatment of various cancers including hematological malignancies. Targeting alkylating moieties to DNA by attachment of a DNA minor groove binding carrier such as distamycin, netropsin, or Hoechst 33252 reduces the loss of active drug due to reaction with other cell components and makes it possible to direct the alkylation both sequence specifically and regiospecifically. We reported the synthesis and structure-activity studies of amidine analogues of alkylating antineoplastic compounds, which appeared to be a new class of cytotoxic minor groove binders and topoisomerase II inhibitors. Another approach to overcome the toxicity of alkylating agents to normal tissue is to construct a prodrug with lower hydrophobicity and cytotoxicity but is preferentially activated in cancer cells. Overexpression of prolidase in some neoplastic cells suggests that the proline analogue of alkylating agents may serve as a prolidase convertible prodrugs. We have compared several aspects of pharmacological actions of proline analogues of chlorambucil and melphalan in breast cancer cells. The results suggest that prolidase could serve as a target enzyme for the selective action of anticancer agents.

Several disubstituted arylene- and chloroambucil-polyamine conjugates were synthesized and evaluated for their ability to target cells via their polyamine transport system (PAT). As compared to the monosubstituted analogues, the disubstituted arylene systems were superior PAT targeting agents. Using a Chinese hamster ovary (CHO) cell line (PAT active) and its CHO-MG mutant (PAT inactive), the series was screened for their PAT targeting ability. The data were expressed as a CHOMG/CHO IC 50 ratio. Indeed, the disubstituted systems gave high IC 50 ratios (e.g., ratio > 2000), which indicated high selectivity for the PAT. The chloroambucil adducts were less toxic than the corresponding arylmethyl compounds. In this regard, having the proper recognition element (i.e., homospermidine) and cytotoxic "cargo" were deemed paramount for successful drug delivery via the PAT.

2-Chloro-2'-deoxyadenosine (cladribine) and chlorambucil are two drugs used in the treatment of lymphoid malignancies. We have synthesized 5'-O-esters of cladribine and its parental nucleoside 2'-deoxyadenosine with chlorambucil (2-chloro-2'-deoxyadenosine-chlorambucil and 2'-deoxyadenosine-chlorambucil, respectively) and compared some properties of the esters with regard to their potential use as antileukemic prodrugs. The 5'-O-ester bond showed no spontaneous hydrolysis at pH 7.4, but was susceptible to hydrolysis by porcine liver esterase and enzymes present in human lymphocyte lysate and blood plasma. Both 2-chloro-2'-deoxyadenosine-chlorambucil and 2'-deoxyadenosine-chlorambucil were taken up more avidly than their parental nucleosides by normal and malignant human lymphoid cells. 2-Chloro-2'-deoxyadenosine-chlorambucil was by an order of magnitude more toxic than 2'-deoxyadenosine-chlorambucil to human leukemic MOLT4 cells in culture. On the other hand, 2-chloro-2'-deoxyadenosine-chlorambucil cytotoxicity did not exceed that of its parental 2-chloro-2'-deoxyadenosine in MOLT4 cells, whereas 2'-deoxyadenosine-chlorambucil was considerably more cytotoxic than free chlorambucil in a variety of myeloid and lymphoid human malignant cell lines. Moreover, acute toxicity of 2'-deoxyadenosine-chlorambucil was lower than that of chlorambucil in mice. In summary, 2'-deoxyadenosine-chlorambucil, but not 2-chloro-2'-deoxyadenosine-chlorambucil, shows promise for clinical utility as a chlorambucil prodrug and thus warrants a more detailed study in vivo.

In order to study the role of the steroidal moiety on the expression of anti-leukemic activity, we synthesized six derivatives of chlorambucil (CHL), and tested them on leukemias P388 and L1210 in vivo and in normal human lymphocytes in vitro. Five of the six tested compounds produced submultiple toxicity, while the measured anti-leukemic potency was significantly increased. The lactamization of the B-steroidal ring rendered the molecules more potent, but the corresponding 7-oxidized derivatives proved better in both leukemias tested. The lactamization of the D-steroidal ring afforded potent compounds, regardless of the configuration of the B-ring. The best among all derivatives contains both chemical modifications and is intended as a promising key molecule that must be further studied. We speculate that in leukemic cells a tumor-specific protein is overexpressed, the steroid has the ability to bind and block this protein from carrying out its normal function, and the drug-protein complex prevents the repair of the adducts. The synthesis, physicochemical and spectroscopic data of these compounds and a modified route for the synthesis of CHL are also reported.

We have studied the effect of modification of the B-steroidal ring to lactamic on the anti-leukemic potency of D-modified and D-non-modified steroidal esters of chlorambucil's active metabolite. The compounds synthesized were studied against leukemias P388 and L1210 after the subsequent estimation of their toxicity in vivo, and for their ability to induce sister chromatid exchanges (SCEs) and to inhibit cell proliferation in normal human lymphocytes in vitro. The in vitro results correlated well, on a molar basis, with the results obtained from the study of the anti-leukemic potency. In a comparative study, the B-lactamic steroidal derivatives proved less active than the 7-oxidized ones against both leukemias. The presence of the -NHCO- group in the B-steroidal ring did not have the same positive effect on the biological action of chlorambucil's active metabolite esters as in the D-lactamic ring. However, this new modification of the B-ring rendered the final esteric derivatives much more toxic, compared with to the corresponding esters with a simple B-ring. This loss of the anti-leukemic specificity, which occurs from the modification of the B-ring, is additional evidence for the role of the steroidal part on the mechanism of action of these promising compounds. This provides support for the notion that the steroidal part of these molecules is not just a simple biological carrier, as has been speculated for many years.

Three steroidal esters with a common alkylating agent (chlorambucil's active metabolite, PHE) and PHE were studied with regard to their genetic activity in human lymphocyte cultures treated in vitro. The cytokinesis block micronucleus assay was used in combination with fluorescence in situ hybridization and the cytosine arabinoside method (ARA-C). The aim of this study was (i) to examine if the modified analogs (EA-72 and SOT-19) of the parent compound (ASE) exerted the same genetic activity with ASE and to correlate the genetic activity with the chemical structure, (ii) to investigate whether these steroidal esters are able to induce excision repairable lesions, through the alkylation of DNA, and (iii) to collect data in order to evaluate the exact role of the steroidal skeleton on the expression of the antileukemic activity. We found that PHE and its steroidal esters are cytotoxic for human lymphocyte cultures, as indicated by the reduction of Cytokinesis Blocked Proliferation Index, PHE being the most cytotoxic molecule. All studied compounds are capable of inducing both chromosome breakage and chromosome delay as indicated by the increased CMN and CMN frequencies. The steroidal derivatives gave reduced genetic activity. The conjugate ketone at the B ring of the steroidal skeleton resulted in decreased genetic activity mainly due to decreased chromosome delay. All studied compounds are capable of inducing DNA excision repair.

A novel amidine analogue of chlorambucil-N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furancarboxamide hydrochloride (AB(1)) and the parent drug were compared for their effects on collagen and DNA biosynthesis in breast cancer MCF-7 cells. IC(50) values for chlorambucil and AB(1) for collagen biosynthesis were found to be about 33 and 13 microM, respectively. The greater potency of AB(1) to suppress collagen synthesis was found to be accompanied by a stronger compared with chlorambucil inhibition of prolidase activity and expression. The phenomenon was related to inhibition of beta(1)-integrin and IGF-I receptor-mediated signaling caused by this compound. The expression of beta(1)-integrin receptor, as well as Src, son of sevenless protein (SOS) and phosphorylated mitogen activated protein (MAP) kinases (MAPK), extracellular-signal-regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)) but not focal adhesion kinase pp125(FAK) (FAK), Shc, and Grb-2 was significantly decreased in cells incubated for 24 h with 10 microM AB(1) compared to the control, whereas in the same conditions chlorambucil did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. Furthermore, AB(1) induced a stronger down-regulation of the expression of IGF-I receptor and evoked a higher antiproliferative effect. During 12 and 24 h of incubation AB(1) decreased DNA biosynthesis by about 33 % and 51 % of the control, whereas chlorambucil decreased it by about 19 % and 35 %, respectively. These data suggest that the amidine analogue of chlorambucil is a stronger inhibitor of protein and DNA synthesis in MCF-7 cells than is the parent drug.

A number of novel cyclic amidine analogs of chlorambucil were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and inhibition of [(3)H]-thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than chlorambucil. The degree to which these compounds inhibited cell growth breast cancer cells was directly correlated to DNA-binding affinity. These studies indicate that cyclic amidine analogs of chlorambucil are a potent catalytic inhibitor of topoisomerase II but not topoisomerase I. The highest degree of DNA binding and cytotoxicity in both MDA-MB-231 and MCF-7 breast cancer cells was observed for the compound, which possess a 4,5-dihydro-1H-imidazol moiety.

A series of amidine analogues of chlorambucil (9-12), where 5-[4-(N-alkylamidino)phenyl]-2-furancarboxamide and the chlorambucil moiety are linked by a NH(CH(2))(2)NH chain, was synthesized and their cytotoxicity has been tested against the growth of human breast cancer MCF-7 cells. Evaluation of the cytotoxicity of compounds 9-12 employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA demonstrated that these conjugates were more active than chlorambucil. Data from the ethidium displacement assay indicated that these compounds bind in the minor groove of DNA and show moderate specificity for AT base pairs. Compounds 9-12 were potent topoisomerase II inhibitors, with 50% inhibitory concentrations (IC(50))ranging from 10 to 40 microM. The cytotoxicity of the compounds 9-12 correlates with their DNA-binding affinities and their relative potency as topoisomerase II inhibitors. Altogether, these data suggest (i) that the cytotoxic activity of compounds 9-12 may be due to the combined effects of alkylation, DNA-minor groove binding, and (ii) that N-(2-aminoethyl)-5-(4-N-alkylamidinophenyl)-2-furancarboxamides (5-8) ligands are suitable linkers that favors DNA targeting by chlorambucil derivatives.

A novel dendritic molecule with nine chlorambucil (CBL) residues on the surface and a maleimide moiety at the core terminus was synthesized using a convergent synthetic methodology. This molecule is ready for attachment to single-chain Fv antibodies (scFvs) to form antibody-multidrug immunoconjugates in an effort to study the relevance of drug/antibody molar ratio and the potency of these drug-antibody immunoconjugates. A monomer and a trimer with a similar structural motif were also prepared for comparative purposes.

Chlorambucilamide derivatives involving chiral glycosyl glycerols derived from D-glucosamine were synthesized in good yield by coupling the chlorambucil moiety to the amino group of omega-amino-(omega-1)-hydroxyalkyl 2-acylamino-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranosides, and subsequent hydrolysis of the benzylidene group. The starting material was easily available from 2-acetamido-2-deoxy-D-glucose. The bonding of 2,3,4,6-tetra-O-pivaloyl-beta-D-galactopyranosylamine to chlorambucil by formation of an amide function is also described.

New carbocylic analogues of distamycin and netropsin with chlorambucil moieties 5-8 have been synthesised. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA. The observed reduced affinity to AT pairs and increased affinity towards GC sequences of the carbocyclic lexitropsins with chlorambucil moiety 5-8 in comparison with netropsin and distamycin was observed and rationalised by means of molecular modelling techniques. All of the compounds 5-8 showed antiproliferative and cytotoxic effects in the standard cell line of the mammalian tumour MCF-7.

We synthesized an proline analogue of chlorambucil (CH-pro) as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidopeptidase--prolidase [E.C.3.4.13.9]. A conjugation of chlorambucil (CH) with proline through an imido-bond resulted in the formation of a good substrate for prolidase. We have compared several aspects of biological actions of CH and its prodrug in cultured normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, in opposition to CH, CH-pro had no inhibitory effect on fibroblast's prolidase activity against the endogenous substrate, glycyl-L-proline. Lower cytotoxicity and a higher antimitotic activity of the prodrug, compared to the free drug, was observed. CH and CH-pro at concentrations of 25 microM led to a 30% and 10%, decrease in cell viability in confluent human skin fibroblasts. IC50 values of CH and CH-pro for DNA synthesis was found to be 30 microM and 7 microM, suggesting higher antimitotic potency of the pro-drug compared to the free drug. CH-pro also evoked lower ability to inhibit collagen biosynthesis in cultured fibroblasts than the free drug. IC50 values of CH and CH-pro for collagen biosynthesis were found at about 15 microM and 30 microM, respectively. Targeting of prolidase as a prodrug-converting enzyme may serve as a novel strategy in pharmacotherapy of various diseases, leading to the increase in therapeutic efficacy and reduction in untoward side effects of antineoplastic agents.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

A 68 year-old-man was first found to have CLL with IgG, kappa monoclonal gammopathy 6 years ago. Bestrabucil (total dose 35,150 mg) was taken orally from August 1989 to December 1989. Etoposide (total dose 23,100 mg) was then orally administered from January 1990 to December 1995. He was then referred to our hospital in January 1996 because of progressive anemia and thrombocytopenia. Peripheral blood showed a WBC of 21,200/microliter with 4% myeloblasts and 79% lymphocytes, Hb 7.9 g/dl and Plt 5 x 10(4)/microliter. The serum level of lysozyme was increased (75.6 micrograms/ml). Bone marrow aspiration disclosed hyper-cellularity with proliferation of the blasts and a monocytoid cell population, which cytochemical studies demonstrated to be of the myelo-monocytic series, thus indicating acute myelogenous leukemia (AML-M4) superimposed on CLL. Surface marker analysis of bone marrow mononuclear cells revealed reactivity for CD 11c, CD13, CD15, CD33, HLA-DR. The karyotype was normal. Southern blot analysis and reverse transcriptase-polymerase chain reaction did not reveal rearrangement of the MLL gene. Complete remission was achieved by chemotherapy consisted of BHAC, idarubicine, 6MP, vincristine and predonisolone. Long-term treatment with oral etoposide may contribute to secondary AML.

Background & Aims: Chemotherapy of hepatocellular carcinomas is hampered by the insufficient accumulation of cytostatic drugs within the tumor cells. The aim of this study was to evaluate the feasibility of therapeutic strategies using antineoplastic agents coupled to bile acids., Methods: Expression of the Na(+)-taurocholate-cotransporting polypeptide (NTCP) was analyzed in six hepatocellular carcinomas and in nonmalignant liver tissue. Uptake of the cytostatic drug [3H]-chlorambucil-taurocholate (S2676) was measured in Xenopus laevis oocytes injected with total messenger RNA (mRNA) from the carcinomas or peritumor tissue or with complementary RNA encoding the NTCP or the organic anion-transporting polypeptide (OATP) of human liver., Results: Expression of hepatocellular carcinoma mRNA in oocytes resulted in mainly Na(+)-dependent uptake of chlorambucil-taurocholate. The level of NTCP mRNA in carcinomas amounted to 56% +/- 27% compared with peritumor tissue. Immunofluorescence studies confirmed the expression of NTCP on the surface of hepatocellular carcinoma cells. OATP expression, determined by immunoblotting, was similar in hepatocellular carcinomas and surrounding liver tissue (n = 3). NTCP mediated Na(+)-dependent uptake of chlorambucil-taurocholate (Michaelis constant, 11 mumol/L), whereas OATP mediated Na(+)-independent uptake., Conclusions: Hepatocellular carcinomas express the Na(+)-dependent bile acid transporter NTCP. Because NTCP mediates high-affinity uptake of chlorambucil-taurocholate, targeting of cytostatic bile acids to hepatocellular carcinomas could become a feasible therapeutic strategy.

New closantel and chlorambucil prodrugs expected to accumulate in the lymphatic system were evaluated on the filaria Molinema dessetae. The prodrugs of closantel had a delayed effect in vitro on the infective larvae compared to the free drug. The closantel prodrugs were less toxic in vivo than closantel itself. The most active prodrug after treatment at 200 mumol/kg by the oral route was the 1,3-dipalmitoyl-2-succinyl-glycerol-closantel. The macrofilaricidal delayed effect of closantel prodrugs was of interest to prevent anaphylactic shock. In vitro, chlorambucil was active on M. dessetae infective larvae with an IC50 of 26 microM. 1,3-Dipalmitoyl-2-chlorambucil-glycerol was slightly active while the addition of a thioether function between the drug and the lymphotropic ligand canceled the activity. However, no activity with chlorambucil and its prodrugs was observed in vivo. The lymphotropism of these prodrugs has now to be verified using comparative pharmacokinetics in serum and lymph to quantify the increase in drug concentration in lymph.

The feasibility to targeting prolidase as an antineoplastic prodrug-converting enzyme has been examined. The synthesis of proline analogue of chlorambucil (well known antineoplastic agent) conjugated through imido-bond (potential target for prolidase action) has been performed. It was found that the product of synthesis, N-[4-[4-(N,N-bis(2-chloroethyl)amino) phenyl]butyryl]-L-proline is insoluble in aqueous solutions but it may be solubilized in methanol. The methanol in 30% concentration reduces catalytic activity of prolidase to 40% of values found in aqueous solution, although it allows in such conditions the measurement of substrate susceptibility to the action of this enzyme. It has been presented that product of synthesis is weakly susceptible to the action of purified prolidase, comparable to the susceptibility of glycyl-L-hydroxyproline. Although insolubility of the proline analogue of chlorambucil in aqueous solutions limit its potential therapeutic value, the presented data suggest that prolidase may have a broader substrate specificity. It suggests that targeting of prolidase as a prodrug-converting enzyme may serve as a novel strategy in therapy of various diseases.

A limitation of the use of chemotherapeutic agents against intracerebral tumors lies on their poor uptake into the central nervous system. An approach to enhance brain delivery is to design agents that are transported into the brain by one of the saturable nutrient carriers of the blood-brain barrier, the highly efficient brain and erythrocyte glucose transporter isoform GLUT1. Since the GLUT1 hexose transporter of the blood-brain barrier is also present on erythrocytes, new compounds designed to be transported by the GLUT1 transporter were studied on human erythrocytes, which represent unique, easily accessible human GLUT1 expressing cells. In this paper we describe the synthesis of four glucose-chlorambucil derivatives, namely methyl 6-O-4[bis(2-chloroethyl)amino]benzenebut anoyl-beta-D-glucopyranosi de (3), 6-O-4-[bis(2-chloroethyl)amino]benzenebu tanoyl-D-glucopyranose (6), methyl 6-[4-[bis(2-chloroethyl)amino]benzenebut anoylamido]-6-deoxy-beta-D-glucopyranoside (9) and 6-[4-[bis(2-chloroethyl)amino]benzenebut anoyl amido]-6-deoxy-D-glucopyranose (10), and the study of their interactions with the GLUT1 transporter of the human erythrocytes. All four compounds were able to inhibit [14C]glucose uptake in a concentration-dependent manner. One of them, compound 6, exhibited an approximately 160-fold higher inhibition of [14C]glucose uptake by the GLUT1 transporter than glucose itself. Compound 6 was also able to inhibit [3H]cytochalasin B binding to erythrocytes with approximately 1000-fold higher efficacy than does glucose. The inhibition of glucose uptake was entirely reversible, indicating that it was not due to alkylation of a nucleophilic group of the hexose transporter. The above results suggested specific interactions of compound 6 with the hexose transporter protein. Uptake studies of [14C]compound 6 indicated, in addition, some non-specific interactions with intact and open erythrocyte membranes: only a small amount of the bound [14C]compound 6 can be displaced by cytochalasin B. Collectively, these findings led us to conclude that the interactions of compound 6 with GLUT1 are presumably that of a non-transported inhibitor.

Immunosuppressants cyclosporin A (CsA), FK506, and KM2210 modulated colony formations of murine hematopoietic progenitor cells. In a 4-h treatment with CsA, 10 micrograms/ml increased the formation of colony-forming units of mixed lineages (CFU-Mix) but decreased the formation of highly proliferative potential colony-forming units (CFU-HPP); 1 microgram/ml of CsA increased the formations of CFU-HPP, CFU-Mix, and colony-forming units of granulocytes/macrophages (CFU-GM); 0.1 microgram of CsA increased the formation of CFU-Mix and burst-forming units of erythroid lineage (BFU-E). Lower doses of CsA appeared to induce an increase in various colony formations. FK506 increased CFU-HPP and CFU-Mix formations at lower doses. Another immunosuppressant, KM2210, increased CFU-HPP and CFU-GM formations but decreased CFU-Mix and BFU-E formations. In a 24-h treatment, 10 micrograms/ml and 1 microgram/ml of CsA inhibited all the colony formations, but 0.1 microgram/ml of CsA increased CFU-Mix, CFU-GM, and BFU-E formations. Similarly, 100 ng/ml and 10 ng/ml of FK506 decreased all the colony formations but 1 ng/ml of FK506 increased CFU-HPP and CFU-GM formations. KM2210 inhibited all the colony formations. These findings showed that lower doses of CsA and FK506 appeared to increase the colony formations, although higher doses of these drugs decreased the colony formations, similar to the findings in a 4-h treatment. On the other hand, KM2210 showed opposing effects on colony formation with 4-h and 24-h treatments.

An in vitro assay method for predicting the hormonal response of primary cultured cells was used in 38 women with breast carcinoma. The response to 17 beta-estradiol (E2) was compared with other hormone treatments, such as tamoxifen (TAM), estracyt (EC) and bestrabucil (BB). The response to the E2-conjugated drugs (EC and BB) and TAM showed a good correlation with the response to E2 (p < 0.01). Coincidental rates of EC and BB response (stimulatory, insensitive, and inhibitory) with E2 response were 47% (16/34) and 53% (16/30) of cases, respectively. Cells showing an inhibitory response to E2 were also inhibited by EC and BB in 60% (6/10) and 100% (8/8) of cases, respectively. In premenopausal women, 28% (5/18) and 43% (6/14) of cases were inhibited by EC and BB, respectively, whereas in postmenopausal women 44% (7/16) and 56% (9/16) of cases showed inhibition with EC and BB, respectively. The inhibitory response to the E2-conjugated drugs was not significantly different between pre- and post-menopausal women. Cells resistant to inhibition by TAM were inhibited by EC and BB, respectively, in 17% (4/24) and 30% (6/20) of cases. These results indicate that E2-conjugated drugs may inhibit the growth of breast cancer cells and that their inhibitory actions might be different from those of TAM.

Cyclosporin A (CsA) inhibited interleukin 2 (IL-2), IL-3, interferon gamma (IFN gamma), GM-CSF and tumor necrosis factor alpha (TNF alpha) mRNA expression in spleen cells stimulated with concavalin A (Con A) when determined by the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. FK506 which has a similar immunosuppressive mechanism to that of CsA also showed the same inhibitory effects except for decreased IL-5 and IL-6 mRNA expression. In contrast, both CsA and FK506 enhanced transforming growth factor beta (TGF beta) and IL-1 beta mRNA expression. Another immunosuppressant KM2210 did not show any inhibitory effects on cytokine gene expression but rather enhanced IL-10, IL-6, TGF beta and IL-1 mRNA expression, thus suggesting that KM2210 has a completely different immunosuppressive mechanism from that of CsA and FK506. Anti-TFG beta 1 antibody abrogated the suppression by KM2210 of BALB/c anti-3H/He mixed lymphocyte reaction (MLR) whereas this antibody did not abrogate the suppression by CsA and FK506 of BALB/c anti-C3H/He MLR. These results indicate that TGF beta is one of the major cytokines in KM2210 immunosuppression, in addition to IL-10, but not in immunosuppression by CsA and FK506.

The potential hypoxia-selective cytotoxin 4-[4'-[N,N-bis(2"-chloroethyl)amino]phenyl]butanoic acid N-oxide (chlorambucil N-oxide, 4) was synthesized and characterized as its hydrochloride salt. This compound was shown to be unstable, decomposing in some organic solvents to the hydroxylamine 4-[4'-[N-(2"-chloroethoxy)-N-(2"-chloroethyl)amino]phenyl]butanoic acid (11) by a mechanism previously demonstrated for aliphatic mustard N-oxides and under aqueous conditions to a more complex mixture, of which the predominant components were the monochloroethyl derivative 7 and formaldehyde. Comparison of NMR spectra showed that a recent published synthesis of 4 in fact resulted in the rearrangement product 11, indicating that recent reported investigations of the hypoxia-selective cytotoxicity and metabolism of chlorambucil N-oxide have examined this rearrangement product rather than 4. In a clonogenic assay, 4 was less cytotoxic against AA8 cells than was chlorambucil, but the effect of oxygen on cytotoxicity was no greater than for chlorambucil itself.

The acute intraperitoneal toxicities of chlorambucil and chlorambucil-spermidine conjugate have been compared, in mice. Both compounds were neurotoxic and also caused a prolonged fall in bodyweight and a depletion of lymphocyte numbers associated with a fall in the total leukocyte count and loss of spleen and thymus weight. Alanine aminotransferase and aspartate aminotransferase activities and blood urea nitrogen concentration were increased at 24 h after conjugate administration, but had returned to normal at 72 h. Chlorambucil significantly decreased blood urea nitrogen concentration for 72 h, but did not affect transferase activity. Tissue concentrations of conjugate were measurable in liver and kidney for 12 days and lung for 5 days after dosing. The toxicity of both compounds was cumulative. In mol/kg, the chlorambucil-spermidine conjugate was 10-fold more toxic than chlorambucil, on the basis of their neurotoxicity, but only 2- to 3-fold more toxic on the basis of their effects on lymphocyte depression. The increased toxicity of the conjugate does not improve its therapeutic index relative to chlorambucil.

Chlorambucil N-oxide (CHLN-O) was synthesized and evaluated for in vitro bioreductive antitumor activity. A time-dependent hypoxic differential was observed when EMT6 cells were exposed to CHLN-O in the presence of rat liver microsomes and reducing equivalents. The cytotoxicity of the N-oxide was potentiated under hypoxia, and augmented further by a combination of low pH and hypoxia. Metabolic studies were also undertaken, which utilized previously described HPLC methodology for the analysis of CHLN-O loss from biological fluids. These demonstrated the requirement for microsomal enzymes and reducing equivalents, and also illustrated the time-dependent manner of CHLN-O loss from isolated microsomal preparations.

The spermidine conjugate of the human carcinogen and cancer therapeutic agent chlorambucil has been evaluated in the mouse bone marrow micronucleus assay. It showed a similar level of activity to that of chlorambucil, indicating that the superior ability of the conjugate to cross-link DNA in vitro is not expressed as enhanced genotoxicity in vivo.

It is known that diffusible trophic factors play an important role in both the normal and cancerous growth regulatory processes of hormone-responsive cells such as are found in the prostate and mammary glands. Consequently, it is important to identify whether the production of such growth factors is affected by administration of therapeutic agents. We examined the effect of bestrabucil, a benzoate of an estradiol-chlorambucil conjugate, on the production of growth factor(s) by Shionogi carcinoma 115 (SC-115) cells, an androgen-responsive cultured cancer cell line. We then investigated whether the inhibitory effect found was specific to bestrabucil, or if it was also produced by a mixture of the 2 compounds, estradiol and chlorambucil. Bioassay employing BALB/3T3 cells demonstrated the presence of two kinds of growth factor in the conditioned medium obtained by culturing SC-115 cells in medium containing 10(-8) M testosterone; these factors could be separated by heparin-sepharose column chromatography using 0.5 M NaCl and 1.1 M NaCl. When the SC-115 cells were cultured in medium containing bestrabucil, at a concentration of 10(-5) M, no growth factor activity was detected in the fraction eluted from the heparin-sepharose column by 1.1 M NaCl. At bestrabucil concentrations of 10(-5)-10(-7) M, concentration-dependent inhibition of growth factor production by SC-115 cells could be demonstrated by 3H-thymidine uptake assay. However, this inhibitory effect could not be demonstrated using only a mixture of estradiol and chlorambucil.(ABSTRACT TRUNCATED AT 250 WORDS)