A subtype prediction score for primary aldosteronism.

Primary aldosteronism (PA) is the most common cause of endocrine hypertension. Although adrenal venous sampling (AVS) is recommended as the gold standard procedure for subtype classification in PA, it is a specialized technique with limited availability. The objective of this study was to develop a scoring system that predicted PA subtype using clinical characteristics. Seventy-one patients with PA were studied. The subjects were diagnosed as having either unilateral (n=32) or bilateral disease (n=39) based on AVS, surgery and/or the postoperative clinical course. Variables associated with laterality in the univariate analysis were entered into multivariable logistic regression models and the regression coefficients were used to construct a subtype prediction score. The diagnostic significance of the score was then evaluated using receiver operating characteristic (ROC) curve analysis. The subtype prediction score was calculated as follows: serum potassium ⩽3.4 mEq l(-1), 2 points; plasma aldosterone concentration ⩾165 pg ml(-1), 3 points; and aldosterone to renin ratio ⩾1000 in a post-captopril challenge test (plasma renin activity in ng ml(-1) h(-1)), 3 points. ROC curve analysis for the ability to discriminate between unilateral and bilateral PA showed that a score of 5 points had 75% sensitivity and 95% specificity, and a score of 3 points had a sensitivity of 97% and a specificity of 59%. The area under the ROC curve was 0.920 (95% confidence interval, 0.859-0.979). Our subtype prediction score could discriminate between unilateral and bilateral PA and is useful for selecting patients who should undergo AVS before surgery.

SU-E-T-557: A Planning Strategy of Volumetric Modulated Arc Therapy with Simultaneous Integrated Boost for Intracranial Stereotactic Radiotherapy.

PURPOSE: The purpose of this planning study was to evaluate the dosimetric effect of dose escalation for intracranial stereotactic radiotherapy by volumetric modulated arc therapy (RapidArc) with simultaneous integrated boost (SIB-VMAT). METHODS: Dynamic conformal arc therapy (DCA), VMAT, and SIB-VMAT plans using Novalis Tx (Varian/BrainLAB) were performed for twenty target volumes in patients with intracranial metastases with median PTV of 16.0 cm3 (range 2.4-35.2 cm3 ). PTV was created with 2 mm expansion from GTV. All plans were generated with a prescribed dose of 35 Gy in 5 fractions to the PTV (D95 = 95%), and dose escalation up to 40 Gy (SIB-VMAT40) and 45 Gy (SIB-VMAT45) was performed only to the PTV-boost (PTV shrunk by 5 mm) for SIB-VMAT. Each plan was compared using conformity parameters. RESULTS: The average Paddick conformity index (CI) was 0.78, 0.90, 0.91, and 0.89 for DCA, VMAT, SIB-VMAT40, and SIB-VMAT45, respectively. The average healthy tissue overdosage factor (HTOF), suggested by SALT was 0.118, 0.006, 0.007, and 0.011 for DCA, VMAT, SIB-VMAT40, and SIB-VMAT45, respectively. The average V30, V20, and V10 of normal brain for VMAT and SIB-VMAT decreased by 3.0 cm3 (range 0.1-8.2 cm3 ), 3.0 cm3 (range 0.1-8.7 cm@@@3@@), and 7.5 cm@@@3@@ (range 0.3-26.2 cm@@@3@@), respectively, compared to DCA depending on the target volume. CONCLUSIONS: SIB-VMAT improved dose conformity to the PTV for intracranial stereotactic radiotherapy, and decreased high and low dose volume of normal brain compared to DCA. SIB-VMAT offers the ability of dose escalation due to high conformity of high dose regions inside the target volume.

Genetic changes in pT2 and pT3 prostate cancer detected by comparative genomic hybridization.

Prostate-specific antigen (PSA) screening has led to a remarkable increase in prostate cancer cases undergoing operative therapy. Over half of patients with locally advanced cancer (>or=pT3) develop rising PSA levels (biochemical failure) within 10 years. It is very difficult to predict which patients will progress rapidly to advanced disease following biochemical failure (BF). Therefore, a more useful prognostic factor is needed to suggest the most appropriate therapies for each patient. To determine chromosomal aberrations, we examined 30 patients with stage pT2 or pT3 primary prostate adenocarcinomas and no metastases (pN0M0) by comparative genomic hybridization (CGH). Laser capture microdissection (LCM) was used to gather cancer cells from frozen prostate specimens. Common chromosomal alterations included losses on 2q23-24, 4q26-28, 6q14-22, 8p12-22 and 13q21-31, as well as gains on 1p32-36, 6p21 and 17q21-22. Losses at 8p12-22 and 13q21-31 were observed more frequently in pT3 than pT2 tumors (P<0.05 and P<0.01, respectively). Losses at 8p12-22 were more frequent in tumors with BF (P<0.05), and those at 13q12-21 were more frequent in tumors with Gleason score (GS) 7 or more than lower GS (P<0.05). These findings suggest that losses of 8p12-22 and 13q21-31 are important determinants of prostate cancer progression.

Evaluation of the basic properties of the BANGkit gel dosimeter.

We evaluated the basic properties of a commercially available BANGkit gel dosimeter, which is a normoxic type of BANG gel. This gel-kit has the same composition as the BANG 3 gel, but is fully oxygenated. To exclude oxygen, oxygen scavenging ascorbic acid and copper sulfate as a catalyst are used. The properties that we examined are the effects of the concentrations of copper sulfate and ascorbic acid on the response, the reproducibility, the long-term stability, the temperature effect at irradiation and the dose-rate effect. In our results, the excellent linear fit of the R2-dose response in a dose range for clinical use and its reproducibility were observed. The precision of a linear fit was preserved for about 3 weeks. The temperature at irradiation showed a significant effect on the dose response. Although the dose-rate dependence in the high-dose range was observed, it was negligible for the clinical dose range up to 270 cGy. In conclusion, this gel dosimeter is thought to be utilizable in clinical practice, while we have to pay attention to the temperature during the entire measurement processes, and additionally there is room for improvement in the linearity and the dose-rate dependence in the high-dose range.

Purpuric adult T-cell leukaemia/lymphoma: expansion of unusual CD4/CD8 double-negative malignant T cells expressing CCR4 but bearing the cytotoxic molecule granzyme B.

A 78-year-old Japanese woman with adult T-cell leukaemia/lymphoma (ATL) presented with an unusual purpuric and erythematous eruption on the face and trunk. Immunohistochemical and flow cytometric analyses showed that the tumour cells were CD4/CD8 double-negative, and expressed CCR4 T-helper (Th) 2 chemokine receptors. Despite these features, the cells aberrantly produced granzyme B, which is a cytotoxic molecule usually produced by CD8(+) cytotoxic T cells, natural killer cells, or occasionally by Th1 cells. In a purpuric lesion, extravasation of erythrocytes was associated with an infiltrate of these cytotoxic tumour cells. Our case suggests phenotypical and functional heterogeneity of tumour cells in ATL, which may be closely related to the clinical appearance of the skin eruption.

The role of epigenetic modifications in retinoic acid receptor beta2 gene expression in human prostate cancers.

The retinoic acid receptor (RAR) beta gene is a putative tumor suppressor gene on chromosome 3p24, where a high incidence of loss of heterozygosity is detected in many types of tumors. Retinoic acid suppresses cancer cell growth through binding to RARs, especially RARbeta, indicating a critical role in mediating anticancer effects. Selective loss or down-regulation of RARbeta mRNA and protein has been reported in prostate cancers (PCas), although the mechanisms remain unclear. We investigated the role of epigenetic modification in RARbeta2 gene silencing. Aberrant methylation was detected in 11 of 14 (79%) primary PCas, 9 of 10 (90%) hormone-refractory PCas, and 2 of 4 (50%) PCa cell lines, but not in any normal prostate samples. Chromatin immunoprecipitation assay showed that all RARbeta2-negative cells (LNCaP, PC3, and DU145) were hypoacetylated at both histones H3 and H4. After exposure to 5-aza-2prime;-deoxycytidine treatment, Trichostatin A and all-trans retinoic acid induced partial demethylation, increased accumulation of acetylated histones, and markedly restored the expression of RARbeta2 in RARbeta2-negative cells. These data suggest that the RARbeta2 gene may be one of the frequently silenced genes by epigenetic modifications in PCa.

Radiation therapy for superficial esophageal cancer: a comparison of radiotherapy methods.

PURPOSE: A comparison of treatment outcomes in response to various methods of radiotherapy for superficial esophageal cancer (SEC) was carried out for a large series of patients. METHODS AND MATERIALS: During the period from March 1987 to November 1998, 147 patients with superficial esophageal cancer received definitive radiation therapy at nine radiotherapy institutions in Japan. Fifty-five patients were treated with external radiation therapy alone, 69 with high-dose-rate intracavitary radiation therapy with or without external radiation therapy, and 23 with low-dose-rate intracavitary radiation therapy and external radiation therapy. RESULTS: The 5-year survival rates for mucosal and submucosal cancer patients were 62% and 42%, respectively. The 5-year cause-specific survival rates for mucosal and submucosal cancer patients were 81% and 64%, respectively (p = 0.013). There was no statistically significant difference in the survival rates for either mucosal or submucosal cancer patients between treatment groups. Metastasis was observed only in submucosal cancer patients. Esophageal ulcers developed only in patients who received intracavitary radiation therapy, and were especially common in patients treated with a fraction size of 5 Gy or more. CONCLUSIONS: The use of intracavitary radiation therapy does not influence the survival or local control rate of SEC. Optimal radiotherapy methods for SEC should be determined by a randomized clinical trial.

Signal therapy for RAS-induced cancers in combination of AG 879 and PP1, specific inhibitors for ErbB2 and Src family kinases, that block PAK activation.

BACKGROUND: Both EGF family ligands and ErbB family receptor kinases act upstream of RAS to induce mitogenesis of normal cells, such as NIH 3T3 fibroblasts. However, oncogenically mutated RAS, such as v-Ha-RAS is constitutively activated and therefore no longer requires these ligands or receptors for its activation. Nevertheless, it up-regulates the expression of these EGF family ligands. To understand the biologic significance of RAS-induced up-regulation of these ligands in both RAS-induced PAK activation and malignant transformation, we have conducted the following studies, based on the previous observations that (1) the N-terminal SH3 domain of PIX selectively binds a Pro-rich domain of 18 amino acids of PAKs, CDC42/Rac-dependent Ser/Thr kinase family, and (2) this specific interaction is essential for both PAK activation and membrane ruffling RESULTS: Using four distinct, cell-permeable, and highly specific inhibitors, namely WR-PAK18, which blocks the PAK-PIX interaction; AG 1478, which inhibits ErbB1 kinase activity; and AG 825 or AG 879, which inhibits ErbB2 kinase activity, we demonstrate that (1) the PAK-PIX interaction is essential for v-Ha-RAS-induced malignant transformation; (2) v-Ha-RAS requires not only ErbB1 but also ErbB2, which are activated through two independent autocrine pathways to induce both the PIX/Rac/CDC42-dependent PAK activation and malignant transformation in vitro; and (3) a combination of AG 879 and the Src family kinase-specific inhibitor PP1 suppresses almost completely the growth of RAS-induced sarcomas in nude mice. CONCLUSION: These findings not only change our conventional view on the role of these RAS-inducible ligands and ErbB family receptors (serving as RAS activators) but also suggest a new avenue for the treatment of RAS-associated cancers by a combination of inhibitors specific for ERbB, Src, or PAK family kinases.

Efficient syntheses of novel C2'-alkylated (+/-)-K252a analogues.

Recent efforts in our laboratories have resulted in a synthetic approach toward C2'-alkylated K252a analogues via extension of a K252a cyclofuranosylation strategy. The bis-indole-N-glycosidic coupling of 6-N-(3,4-dimethoxybenzyl)-staurosporinone (21) with a number of highly functionalized carbohydrates has given access to previously unattainable, biologically relevant analogues.

Radiotherapy combined with transcatheter arterial infusion of cisplatin versus oral fluoropyrimidine anticancer agent for locally advanced carcinoma of the uterine cervix: a prospective follow-up study.

We randomized patients with locally advanced cervical cancer to receive radiotherapy combined with transcatheter arterial infusion (TAI) of cisplatin or oral fluoropyrimidine anticancer agents, and compared the prognosis by a prospective follow-up study. Sixty patients were studied who completed their planned radiation therapy with chemotherapy at the Department of Obstetrics and Gynecology of Hiroshima University Hospital between January 1991 and December 1998. Patients were randomly assigned to receive (A) radiotherapy with TAI of 120 mg/body cisplatin twice a month at the interval of 4 weeks or (B) radiotherapy with 200 mg/day oral 5-FU or UFT every day. In both groups, radiotherapy is routinely 50 Gy of external beam irradiation to the whole pelvis and 18-20 Gy (point A dose) of intracavitary irradiation using a remote after loading system (RALS). Serious adverse reactions interfering with treatment did not appear in either group. The effective histologic response was 28/32 (87.5%) in group A and 25/28 (89.3%) in group B. The median follow-up period were 28.3 months and 25.4 months in group A and B, respectively. There was no significant difference in the overall survival and disease-free survival rates for all patients, clinical stage III and squamous cell carcinoma. We could not conclude that radiotherapy with TAI of cisplatin achieved superior therapeutic efficacy in locally advanced cervical cancer. To improve the therapeutic effects, it is important to establish a new cisplatin-containing chemoradiotherapy regimen.

A practical preparation of methyl 2-methoxy-6-methylaminopyridine-3-carboxylate from 2,6-dichloro-3-trifluoromethylpyridine.

An effective and practical synthetic route to methyl 2-methoxy-6-methylaminopyridine-3-carboxylate (7), the key intermediate of 5-bromo-2-methoxy-6-methylaminopyridine-3-carboxylic acid (1), from 2,6-dichloro-3-trifluoromethylpyridine (12) was undertaken. Process improvements were highlighted by regioselectivity of 12 with a nitrogen nucleophile and conversion of the 3-trifluoromethyl group into the methoxycarbonyl group. The reaction of 12 with N-benzylmethylamine provided the 6-(N-benzyl-N-methyl)aminopyridine 26a and the regioisomer 26b in >98:<2 ratio in a quantitative yield. Treatment of 2-methoxy-6-methylamino-3-trifluoropyridine (14a) with a large excess of sodium methoxide followed by acid hydrolysis gave the pyridine-3-carboxylic ester 7 in an excellent yield. The potential application of this reaction is also described.

Mycobacterium tuberculosis infection within Warthin's tumor: report of two cases.

We report two patients with Warthin's tumor who were also infected with Mycobacterium tuberculosis. Case 1 was a 75-year-old woman with Warthin's tumor and multiple small epithelioid granulomas with caseous necrosis involving the submandibular gland. This patient died of tuberculous meningitis 4 months after biopsy. Case 2 was a 78-year-old man with a 10-year history of a parotid mass which had enlarged rapidly over 2 months. Surgical excision revealed Warthin's tumor and epithelioid granulomas involving the left parotid gland. DNA extracted from paraffin sections was amplified by nested polymerase chain reaction (PCR) with primer sets for the mycobacterial 65-KDa antigen gene. Restriction enzyme digestion of the PCR products could differentiate Mycobacterium tuberculosis from other mycobacteria in both cases. Although the histogenesis of lymphoid components of Warthin's tumor is controversial, the frequent prevalence of inflammation or necrosis and our present findings suggest these components have a similar behavior to regional lymph nodes.

An anti-Ras cancer potential of PP1, an inhibitor specific for Src family kinases: in vitro and in vivo studies.

BACKGROUND: We previously found that both PAK, a Rac/CDC42-activated Ser/Thr kinase, and its binding partner PIX are required for malignant transformation caused by oncogenic Ras mutants, such as v-Ha-Ras. Furthermore, oncogenic Ras requires an autocrine pathway to activate PAK. This pathway involves at least two distinct receptor kinases: EGF receptor (ErbB1) and ErbB2. Interestingly, both of these kinases are known to activate Src family kinases that phosphorylate CAT, another binding partner of PIX. PURPOSE: The major aim of this study was to determine whether Src family kinases are required for both Ras-induced PAK activation and malignant transformation. For this purpose, we used PP1, an inhibitor specific for Src family kinases, which does not inhibit either EGF receptor or ErbB2. METHODS AND RESULTS: We studied the effect of PP1 on the anchorage-dependent growth of normal and v-Ha-Ras transformed NIH 3T3 fibroblasts, PAK activation and anchorage-independent growth of Ras transformants, and development of Ras-induced sarcomas in nude mice. We found that PP1 (10 nM) strongly inhibits PAK activity in Ras transformants. PP1 at this concentration is known to inhibit c-Fyn kinase, but not c-Src kinase, and none of the three known Src family kinases (c-Src, c-Fyn, and c-Yes) expressed in fibroblasts is activated by v-Ha-Ras. Thus, it is most likely that the primary target of this drug is an as yet unidentified Ras-activated Tyr (Y) kinase or kinases, which we call "Ray." Although PP1 has no effect on their anchorage-dependent growth, it significantly inhibits their anchorage-independent growth in soft agar, as well as a rapid growth of Ras-induced sarcomas in mice. CONCLUSION: Like EGF receptor and ErbB2, a member of Src family kinases (most likely a new Src-related kinase called "Ray") is essential for the Ras-induced activation of PAK and the malignant transformation both in vitro and in vivo. These findings suggest that PP1 and other inhibitors specific for Src family kinases are potentially useful for the treatment of Ras-associated cancers.

Autocrine and paracrine Müllerian inhibiting substance hormone signaling in reproduction.

Members of the transforming growth factor beta (TGFbeta) superfamily are polypeptide growth factors that exhibit diverse effects on normal cell growth, adhesion, mesenchymal-epithelial interactions, cell differentiation, and programmed cell death. This chapter will discuss the work of ourselves and others on one member of this large superfamily, Müllerian inhibiting substance (MIS, or anti-Müllerian hormone, AMH) and its role in reproductive tract development and the adult gonad. Using recombinant MIS protein, it is possible to begin unraveling the molecular mechanism of duct involution in the embryo. Our recent results suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions. In addition to the developmental effects of MIS in secondary sexual differentiation, expression studies of the MIS ligand and the MIS type II receptor (MISIIR) suggest a potential regulatory role for MIS in adult germ cell maturation and gonadal function. Recent data from others suggest that MIS may act in a paracrine manner to block differentiation of interstitial cells of the adult gonad by repressing all or some steps of steroidogenesis. Our studies are highly suggestive of direct repression of steroidogenic enzyme gene expression by activation of the MIS signaling pathway. Thus, for the first time, an opportunity to define fully target genes and components of the MIS signaling pathway may be possible.

Treatment of ras-induced cancers by the F-actin-bundling drug MKT-077.

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.

Expression of MAT1/PEA-15 mRNA isoforms during physiological and neoplastic changes in the mouse mammary gland.

MAT1 is a novel transforming gene which was cloned from a mouse mammary tumor induced by N-methyl-N-nitrosourea in vitro in the presence of lithium as a mitogen. Later, it was found to be identical to the 3' untranslated region (UTR) of the 2.5 kb isoform of PEA-15 (phosphoprotein enriched in astrocytes-15 kDa). We re-cloned MAT1/PEA-15 cDNAs and showed 2.5, 2.0 and 1.8 kb isoforms and confirmed MAT1 localization as reported. The 2.0 and 1.8 kb isoforms were produced by alternative splicing and alternative polyadenylation at the 3' UTR, respectively. To analyze the role of MAT1/PEA-15, we examined the expression of MAT1/PEA-15 mRNA in normal mammary tissues and in mammary tumors. The mammary gland during pregnancy, lactation and weaning showed weak but stable expression. Compared with normal mammary gland, mammary tumors showed stronger expression. Aberrant expression of MAT1/PEA-15 isoforms was found in mouse mammary epithelial cell lines, FSK7 and TM6, which lost the 2.5/2.0 and 2.5 kb isoforms, respectively. In contrast to other oncogenes like c-myc, MAT1/PEA-15 mRNA was extremely stable after actinomycin D and cycloheximide treatments suggesting that other protein expression is prerequisite for degradation of MAT1/PEA-15 mRNA. It evoked the possibility of the 3' UTR of MAT1/PEA-15 (designated as MAT1-T) as a riboregulator in mammary tumorigenesis and necessity for further analysis of human breast cancers as well as mouse mammary tumors.

A preliminary outcome analysis of the Patterns of Care Study in Japan for esophageal cancer patients with special reference to age: non surgery group.

BACKGROUND: The Patterns of Care Study (PCS) was imported to Japan from the United States in July 1996. A preliminary outcome analysis of the PCS for esophageal cancer patients in Japan was made with special reference to age, because the elderly population is rapidly increasing in Japan. PATIENTS AND METHODS: From July 1996 to February 1998, external PCS audits were performed for 37 institutions nationwide and detailed information of 561 esophageal cancer patients treated during the period 1992-1994 was collected by using the fifth PCS data format developed in the United States. This format was provided courtesy of the American College of Radiology. For this study, patients who had not undergone surgery (n = 336) were selected. The patients were classified into three age groups: < 65 years old (n = 119), between 65 and 74 years (n = 93), and 75 years or older (n =123). Cox's proportional hazards model was used for the statistical analysis, with survival, acute/subacute complication and late complication of grade 3 or more based on RTOG criteria, as the endpoints. RESULTS: Significant prognostic factors for the entire non-surgery group were Karnofsky Performance Status (KPS) (p = 0.0007), stage (p = 0.0001), and external irradiation dose (p = 0.0001). For the younger group, KPS (p = 0.0004), stage (p = 0.0197), and utilization of brachytherapy (p = 0.0010) were significant, while for the intermediate age group it was KPS (p = 0. 0027), history of pulmonary disease (p = 0.0339), stage (p = 0.0001), and external dose (p = 0.0001), and for the elderly group, stage (p = 0.0001) and external irradiation dose (p = 0.0224) were significant. Significant risk factors for complications for the entire group were stage (p = 0.0411), external dose (p = 0.0163), and stratification of institution (academic vs. nonacademic) (p = 0. 0114). Significant risk factors for the younger group were history of pulmonary disease (p = 0.0495) and external dose (p = 0.0037), and the other age groups showed no significant risk factors. CONCLUSION: Age was not a significant prognostic or risk factor for esophageal cancer patients in the non-surgery group treated with radiation therapy. Therefore, radiation therapy represented an important treatment modality for the elderly as well as for the younger esophageal cancer patients. External dose was a treatment-related prognostic factor for the elderly as well as for the intermediate age group.

An efficient synthesis of 5-bromo-2-methoxy-6-methylaminopyridine-3carboxylic acid.

An efficient synthesis of 5-bromo-2-methoxy-6-methylaminopyridine-3-carboxylic acid (1), a carboxylic acid moiety of a potent dopamine D2 and D3 and serotonin-3 (5-HT3) receptors antagonist, (R)-5-bromo-N-(1-ethyl-4-methylhexahydro-1 ,4-diazepin-6-yl)-2-methoxy-6-methylaminopyridine-3-carboxamide, is described. Reaction of methyl 2,6-difluoropyridine-3-carboxylate (12) with methylamine in EtOH at -25 degrees C gave a mixture of methyl 2-fluoro-6-methylaminopyridine-3-carboxylate (13) and the regioisomer 14 in a ratio of 57 : 43. On the other hand, reaction of 12 and methyl 2,6-dichloropyridine-3-carboxylate (16) with sodium methoxide in tetrahydrofuran (THF) and CH2Cl2 provided the 2-methoxypyridine-3-carboxylic esters 20 and 23, respectively, as main products. Similar reaction of 16 in N,N-dimethylformamide (DMF) and MeOH proved to be highly regioselective for the 6-position. A much greater regioselectivity for substitution at the 6-position (>97%) was observed when 16 was treated with 4-methylbenzenethiolate anion in DMF (quantitative yield). After methoxylation of methyl 2-chloro-6-(4-methylbenzenethio)pyridine-3-carboxylate (25b) and successive oxidation of the 6-benzenethio moiety, nucleophilic substitution of the sulfoxide derivative 28 with methylamine gave the 6-methylamino derivative 8. Finally, bromination of 8 and alkaline hydrolysis produced the desired product 1 in an overall yield of 67%.