Effect Of Urinary Kidney Injury Molecule-1 Levels On Short-Term Prognosis Of Chronic Heart Failure.

Objectives: To explore the influential elements of urinary kidney injury molecule-1 levels in chronic heart failure, and to judge its ability to predict 90-day rehospitalisation. METHODS: The cross-sectional case-control study was conducted from November 2020 to April 2021, at Hanzhong Central Hospital, China, and comprised adult patients having chronic heart failure with normal renal function in group A and healthy subjects in control group B. Patients in group A received anti-heart failure therapy for 1 week in hospital and were followed up for 90 days after discharge. Blood pressure (BP), kidney injury molecule-1, creatinine and serum pro- B-type natriuretic peptide levels were evaluated at baseline and 1 week after treatment in group A, while the samples were collected only at baseline in the control group B. Data was analysed using SPSS 22. RESULTS: Of the 102 subjects, 68(66.6%) were in group A; 44(64.7%) males and 24 (35.3%) females with mean age 62.38±9.51 years. The remaining 34(33.3%) subjects were in group B; 21(61.7%) males and 13(38.2%) females with mean age vs. 58.82±8.11 years. The urinary kidney injury molecule-1 level in group A was essentially on the increase compared to group B (p<0.05). After 1 week of treatment, the kidney injury molecule-1 level decreased compared to the baseline value in group A (p<0.05). Diastolic blood pressure and pro-B-type natriuretic peptide were the determinants of urinary kidney injury molecule-1 level, and urinary kidney injury molecule-1 level before discharge was significantly associated with rehospitalisation within 90 days (p<0.05). CONCLUSIONS: Urinary kidney injury molecule-1 level before discharge was a significant predictor of rehospitalisation within 90 days, and diastolic blood pressure and pro-B-type natriuretic peptide levels were the influencing factors of urinary kidney injury molecule-1. Also, urinary kidney injury molecule-1 levels were significantly raised in chronic heart failure.

Experimental Study on Solidification Characteristics of Sessile Urine Droplets on a Horizontal Cold Plate Surface under Natural Convection.

As the human excreta, urine is often used as one of the test materials in medical research due to its composition and content directly reflecting the health status of the body. Considering that the substances in urine may show different effects on its freezing process, solidification characteristics of sessile urine droplets on a horizontal cold plate surface under natural convection were experimentally investigated by comparing with those of water droplets under same conditions. To make the conclusion analysis more reasonable, the urine of a human without any diseases, especially metabolic diseases, was treated and used. The characteristics include nucleation location, dynamic variation of droplet color, and temperatures at different heights inside the droplet, and so forth. It was found that, similar to that of a water droplet, the solidification process of a urine droplet also experiences the following four stages: supercooling, recalescence, freezing, and cooling, in chronological order. Differently, the urine droplet changes from transparent to blur white at the supercooling stage due to the precipitation of inorganic salts. For nucleation locations, 46.67% cases are at the bottom, while others are at the top and middle of urine droplets. For a 10 µL droplet on a surface of -30 °C, urine has a 0.95 s freezing duration shorter than water, and a 5.31 °C lower phase-transition temperature. Results of this study are expected to reflect the content of substances in urine and thus provide references for urinalysis of patients with metabolic diseases.

Aromatic Heterocycles as Productive Dienophiles in the Inverse Electron-Demand Diels-Alder Reactions of 1,3,5-Triazines.

Heterocycles are often found as the structural nucleus in natural products and biological active compounds. Consequently, research toward the discovery and development of novel and efficient synthetic methodologies is of constant interest to organic chemists. Diels-Alder reactions are powerful at forming multiple bonds simultaneously, often with stereoselectivity, and thus are one of the most widely used synthetic methodologies in organic syntheses. Inverse electron-demand Diels-Alder (IEDDA) reactions, a subclass of Diels-Alder reactions, have been developed for the efficient synthesis of various heterocycles, with 1,3,5-triazines used as azadienes. The initial 1,3,5-triazine IEDDA reactions mostly included nonaromatic, electron-rich species such as vinyl ethers, enamines, ynamines, and amidines as dienophiles, producing in high yields pyrimidine derivatives with excellent regioselectivity. We hypothesized that some electron-rich aromatic heterocycles could be studied as potential dienophiles for 1,3,5-triazine IEDDA reactions; 5-aminopyrazoles proved to be productive dienophiles leading to high yields of pyrazolopyrimidines. Subsequently, many studies were reported to investigate the mechanism and scope of this new type of IEDDA reaction. Mechanistically, this new type of IEDDA reaction is quite interesting since it entails two aromatic compounds proceeding through a perceived high energy nonaromatic transition state, leading to a new aromatic compound, a counterintuitive process. Both theoretical and experimental studies provide key insights to this reaction mechanism, with learnings from these studies possibly stimulating unconventional thinking in other areas. Theoretical calculations of these cascade reactions of amino-substituted heterocycles with 1,3,5-triazines indicate that the reactions occur in a stepwise fashion via a highly polar zwitterionic intermediate; elimination of ammonia from IEDDA adducts and subsequent retro Diels-Alder reaction drive the reaction toward the fully aromatized IEDDA products. This amino substituent is critical in determining the regioselectivity and driving the cascade reactions to completion. With regard to reaction scope, many amino-heterocycles such as pyrroles, imidazoles, furans, thiophenes, and indoles all proved to be productive dienophiles for this new IEDDA reaction, leading to various fused-pyrimidines in a single step with moderate to high yields and high regioselectivity. Application of this new IEDDA reaction with 1,3,5-triazines was reported to lead to interesting heterocyclic compounds such as nucleoside natural product nebularine and analogues, as well as fluorine-containing fused pyrimidines with potential for biological activities.Herein, the scope of various aromatic heterocycles as dienophiles in the 1,3,5-triazine IEDDA reaction is reviewed. Moreover, both experimental and theoretical studies of the mechanisms for this interesting cascade IEDDA reaction are discussed. Finally, applications of this new type (aromatic heterocycles as dienophiles with 1,3,5-triazines as azadienes) of IEDDA reaction are also covered.

Reaction of Aldehydes/Ketones with Electron-Deficient 1,3,5-Triazines Leading to Functionalized Pyrimidines as Diels-Alder/Retro-Diels-Alder Reaction Products: Reaction Development and Mechanistic Studies.

Catalytic inverse electron demand Diels-Alder (IEDDA) reactions of heterocyclic aza-dienes are rarely reported since highly reactive and electron-rich dienophiles are often found not compatible with strong acids such as Lewis acids. Herein, we disclose that TFA-catalyzed reactions of electron-deficient 1,3,5-triazines and electron-deficient aldehydes/ketones can take place. These reactions led to highly functionalized pyrimidines as products in fair to good yields. The reaction mechanism was carefully studied by the combination of experimental and computational studies. The reactions involve a cascade of stepwise inverse electron demand hetero-Diels-Alder (ihDA) reactions, followed by retro-Diels-Alder (rDA) reactions and elimination of water. An acid was required for both ihDA and rDA reactions. This mechanism was further verified by comparing the relative reactivity of aldehydes/ketones and their corresponding vinyl ethers in the current reaction system.

[Effect of bazedoxifene on endometriosis in a rat model].

OBJECTIVE: To investigate the therapeutic mechanism of bazedoxifene, the third-generation selective ER modulator (SERM), on endometriosis lesions in a rat model. METHODS: Endometriosis was induced by transplanting pieces of endometrium from other syngeneic rats that were as donors onto the subcutaneous of other unmated female rats. The rats with successful ectopic implants were divided into two groups: control group (n=10) and bazedoxifene group (n=10). The macroscopic morphology, volume, histopathology of ectopic implant and rats uterine wet weight were determined before and after the treatment. Expression of proliferation cell nuclear antigen (PCNA), ER and PR in the eutopic endometrium and endometriosis lesions detected by immunohistochemistry in the two groups. RESULTS: (1) The gross morphology and histological changes of endometriosis lesions in rats after treatment: compared with the control group, it was obviously depauperated and had more less glands and blood vessels in the stroma. (2) The change of rats' weight, the volume of endometriosis lesion before and after treatment and rats uterine wet weigh after treatment respectively in the control group and the bazedoxifene group: rats' weight were respectively before treatment: (201±17) g, (202±18) g, that were respectively after treatment: (266±16) g, (261±16) g, which showed no significant difference between two groups before and after treatment (P>0.05). The volume of ectopic implant before treatment were respectively (85±17) mm3, (85±12) mm3, and showed no significant difference between two groups; that were respectively (48±11) mm3, (24±9) mm3 afte rtreatment, which was significantly decreased compared with the control group (P<0.05). Rats uterine wet weight after treatment were respectively: (0.77±0.16) g, (0.45±0.18) g, and was significantly reduced compared with the control group (P<0.05). (3) The protein expression levels of PCNA, ER and PR in the endometriosis lesions after treatment were respectively 0.282±0.044, 0.51±0.06, 0.49±0.05 in the control group, 0.191±0.020, 0.23±0.03, 0.48±0.06 in the bazedoxifene group; that in eutopic endometrium were respectively 0.369±0.081, 0.56±0.08, 0.56±0.10 in the control group, 0.211±0.037, 0.27±0.05, 0.54±0.08 in the bazedoxifene group; the protein expression levels of PCNA and ER in endometriosis lesions and the eutopic endometrium were significantly decreased in the bazedoxifene group compared with the control group (P<0.05), but the protein levels of PR in endometriosis lesionsand and the eutopic endometrium were not significantly altered by treatment (P>0.05). CONCLUSION: Bazedoxifene could obviously reduce the size of endometriosis lesions, the mechanism may be related with suppressing estrogen-induced proliferation, the expression of ER and direct ER antagonism by this SERM.

Efficacy and safety of laparoscopic vaginoplasty with peritoneal flaps and cervicoplasty in patients with congenital cervical and complete vaginal atresia: a pilot study.

Background: Hysterectomy places a considerable physical and mental burden on young female patients with congenital cervical and complete vaginal atresia. Thus, it is necessary to develop a method to detach the obstruction and simultaneously preserve the vagina and uterus in these patients. This study sought to evaluate the efficacy and safety of laparoscopic vaginoplasty using peritoneal flaps and cervicoplasty in patients with congenital cervical and complete vaginal atresia. Methods: Between April 2013 and June 2022, nine patients with congenital cervical and complete vaginal atresia at Henan Provincial People's Hospital were enrolled in this prospective study. All patients were treated with laparoscopic vaginoplasty using peritoneal flaps and cervicoplasty. Baseline clinical data (e.g., age and uterus size) were collected. The surgical success rate and adverse events were assessed. Results: The nine enrolled patients had a median age of 15.0 [interquartile range (IQR), 14.0-18.0] years, and five of these patients had pelvic adhesions. The surgeries were successful in all (9/9) patients, with the vagina, uterus, and a normal menstrual cycle being preserved. After a median follow-up duration of 48 months, the neovaginas had a median length of 7.5 cm. Postoperative complications occurred in three of patients and were cured with the appropriate treatment. The five married patients reported being satisfied with their sex life. Conclusions: The study preliminarily demonstrated the efficacy of laparoscopic vaginoplasty using peritoneal flaps and cervicoplasty in patients with congenital cervical and complete vaginal atresia. However, due to the small sample size, lack of a control group, and relatively high incidence of adverse events, further studies are still needed to verify these results. Regardless, our findings establish an approach for preserving both the vagina and uterus for patients with congenital cervical and complete vaginal atresia.

Functional analysis of two mutation sites in the OCA2 gene.

To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA and protein levels via the use of recombinant carriers in vitro. Whole-exome sequencing (WES) and Sanger sequencing were used to analyse the pathogenic genes of the child and validate the mutations in the parents. pEGFP and phage vectors carrying wild-type and mutant OCA2 were constructed using the coding DNA sequence (CDS) of the whole gene-synthesized OCA2 as a template and transfected into HEK293T cells, after which expression analysis was performed. The child in this study was born with white skin, hair, eyelashes, and eyebrows and exhibited nystagmus. Genetic analysis indicated that the child carried two heterozygous mutations: c.1079C > T (p.Ser360Phe) of maternal origin and c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) of paternal origin, conforming to an autosomal recessive inheritance pattern. In vitro analysis showed that the expression of the c.1079C > T (p.Ser360Phe) mutant did not significantly change at the mRNA level but did increase at the protein level, suggesting that the mutation may lead to enhanced protein stability, and the c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) mutation resulted in the loss of three amino acids in exon 10, producing a truncated protein. In vitro expression analysis also revealed that the expression of the mutant gene was significantly downregulated at both the mRNA and protein levels, suggesting that the mutation can simultaneously produce truncated proteins and lead to protein degradation. This case study enriches the phenotypic spectrum of OCA2 gene disease. In vitro expression analysis confirmed that both mutations affect protein expression, providing a theoretical basis for analysing the pathogenicity of these two mutations.

Retraction: efficacy and safety of laparoscopic vaginoplasty using the peritoneal flap and cervicoplasty in patients with congenital cervical and complete vaginal atresia: a pilot study.

[This retracts the article DOI: 10.21037/atm-23-217.].

Efficacy and safety of laparoscopic vaginoplasty using the peritoneal flap and cervicoplasty in patients with congenital cervical and complete vaginal atresia: a pilot study.

Background: Hysterectomy places a huge physical and mental burden on young female patients with congenital cervical and complete vaginal atresia. Thus, it is necessary to develop a method to detach the obstruction and simultaneously preserve the vagina and uterus in these patients. This study sought to evaluate the efficacy and safety of laparoscopic vaginoplasty using the peritoneal flap and cervicoplasty in patients with congenital cervical and complete vaginal atresia. Methods: Between April 2013 and June 2022, 9 patients with congenital cervical and complete vaginal atresia at Henan Provincial People's Hospital were enrolled in this prospective study. All the patients were treated with laparoscopic vaginoplasty using the peritoneal flap and cervicoplasty. Baseline clinical features (such as age, uterus size, etc.) were collected. The surgical success rate and adverse events were assessed. Results: The 9 enrolled patients had a median [interquartile range (IQR)] age of 15.0 (14.0-18.0) years, and 5/9 patients presented with pelvic adhesions. The surgeries were successful in all (9/9) patients, who preserved their vagina and uterus with a normal menstrual cycle. After a median follow-up duration of 48 months, the neovagina had a median length of 7.5 cm. Post-surgical complications occurred in 3/9 patients, which were cured by an appropriate treatment. The 5/9 married patients reported being satisfied with their sexual life. Conclusions: Even though the current study preliminary exhibits the efficiency of laparoscopic vaginoplasty using the peritoneal flap and cervicoplasty in patients with congenital cervical and complete vaginal atresia, due to the small sample size, lack of a control group, and relatively high incidence of the adverse events, further studies are still needed to verify the current findings. The current study put forward a further direction for preserving the vagina and uterus simultaneously for those patients with congenital cervical and complete vaginal atresia.

Synthesis of novel 4H-pyrimido[1,6-a]pyrimidines via a one-pot three-component condensation.

Highly substituted novel 4H-pyrimido[1,6-a] pyrimidines were prepared by a trifluoromethanesulfonic acid catalyzed one-pot three-component condensation of 4-aminopyrimidines, aldehydes, and ß-ketoesters. A preliminary feasibility study was undertaken on these compounds, to assess the potential production of a library of further diversified compounds by nucleophilic replacement of Cl(R(1)) or by reaction of electrophiles with the NH(2)(R(2)) group.

Synthesis of highly substituted 2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-ones from 4,6-dichloro-5-formylpyrimidine, amines and aldehydes.

A practical strategy was developed for the preparation of highly substituted 2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-ones from 4,6-dichloro-5-formylpyrimidine, primary amines, and aldehydes. The key step for this synthesis entails a cyclization reaction involving an intramolecular amide addition to an iminium intermediate formed in situ from 4-amino-pyrimidine-5-carboxamide 2 and aldehydes to form the pyrimido[4,5-d]pyrimidine core with a strategically placed 5-Cl group for further derivatization. The utility of this methodology was demonstrated through the preparation of a 27-membered library of representative 2,3-dihydropyrimido[4,5-d]pyrimidin-4(1H)-ones in moderate to good yields.

Low-molecular-weight heparin versus unfractionated heparin in acute ST-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention with drug-eluting stents.

BACKGROUND: Whether low-molecular-weight heparin (LMWH) is superior to unfractionated heparin (UFH) in acute ST-segment elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PCI) with drug-eluting stents (DESs) remains unclear. METHODS: A total of 3,372 STEMI patients who underwent primary PCI with DESs received either LMWH (n = 1,531 patients, subcutaneous enoxaparin 1 mg/kg, bid for 3-5 days plus reduced dose of UFH [50 U/kg] during PCI) or UFH alone (n = 1,841 patients, intravenous bolus injection of 5,000 U, followed by 24,000 U/d infusion for at least 48 hours). The bleeding events and clinical outcomes during in-hospital and at 8 months were compared. RESULTS: The incidences of major and minor bleeding events were similar between the 2 groups. Multivariable Cox regression analysis showed that LMWH group had lower incidences of cardiac death (adjusted odds ratio [OR] 0.55, 95% CI 0.39-0.77, P < .001), total death (adjusted OR 0.50, 95% CI 0.37-0.68, P < .001), and total major adverse cardiac events (adjusted OR 0.77, 95% CI 0.62-0.95, P = .017) at 8 months as compared with UFH group. Similar results were obtained across different subgroups including different DESs, age, and sex. CONCLUSIONS: The LMWH enoxaparin combined with reduced dose of UFH (50 U/kg) administration as an adjunctive antithrombotic therapy in STEMI patients undergoing primary PCI with DESs seems to be safe and efficacious. However, randomized clinical trials are needed to confirm this conclusion.

Stereochemistry as a tool in deciphering the processes of a tandem iminium cyclization and Smiles rearrangement.

To understand the detailed mechanism of a recently reported tandem iminium cyclization and Smiles rearrangement, the reaction processes of a chiral substrate were investigated by monitoring its stereochemical courses. Under the tandem reaction conditions, chiral aldehyde 1 derived from l-prolinol led to two surprising results. First, the iminium cyclization gave a diastereomeric mixture with the cis-configured product as the predominant one. Second, Smiles rearrangement of both cis- and trans-2 led to the same product 3a directly derived from the trans isomer. The former was rationalized by the postulation of a Cram's chelate transition state leading to the cis product as kinetically favored. The latter was due to the equilibration between the trans/cis pair involving a carbocation intermediate and the steric hindrance, which prevented the cis isomer from undergoing the intramolecular nucleophilic substitution. This hypothesis was further supported by the results of a competition experiment in which the addition of 1 equiv of p-methoxyaniline in the rearrangement step led to a significant amount of anilinyl-exchanged rearrangement product.

Synthesis of novel 8,9-dihydro-5H-pyrimido[4,5-e][1,4]diazepin-7(6H)-ones.

Practical and efficient methods have been developed for the synthesis of 4,6,8,9-tetrasubstituted 8,9-dihydro-5H-pyrimido[4,5-e][1,4]diazepin-7(6H)-ones from 4,6-dicholoropyrimidine aldehyde, N-substituted amino acid esters, and amines in five steps. This synthetic strategy is based on suitably substituted pyrimidines as bis-electrophilic species reacting with various amines to construct the pyrimido[4,5-e][1,4]diazepine core with a strategically anchored functional group for further derivatization. The utility of this methodology was demonstrated through the preparation of a 33-membered library of representative 8,9-dihydro-5H-pyrimido[4,5-e][1,4]diazepin-7(6H)-ones in good to excellent yields.

Synthesis of hexahydrobenzo[b]pyrimido[4,5-h][1,6]naphthyridines via an intramolecular hetero-Diels-Alder reaction.

Method development was completed for a strategy to access a novel pyrimidine-fused heterocyclic scaffold. The key step for this synthetic route entails an intramolecular inverse electron demand hetero-Diels-Alder reaction of imines or iminiums formed in situ from allylaminopyrimidinealdehydes 3 and anilines. The reactions provided exclusively cis-configuration products 6. Products 6 were readily precipitated in the reaction solution in good to excellent yields. Further transformations of the phenylthio group were demonstrated by an oxidation and subsequent nucleophilic substitution sequence. The synthetic strategy provides an efficient way to access libraries of the tetracyclic pyrimidine-fused heterocycles that can be explored for potential pharmaceutical or biological activities.

Clinical characteristics and mid-term outcomes of acute myocardial infarction patients with prior cerebrovascular disease in an Asian population: Lessons from the Korea Acute Myocardial Infarction Registry.

1. The aim of the present study was to evaluated the impact of prior cerebrovascular disease (CVD) on the clinical characteristics and mid-term clinical outcomes of patients with acute myocardial infarction (AMI) in the era of drug-eluting stents. 2. Data from 12 914 patients with acute myocardial infarction who were enrolled in the Korea Acute Myocardial Infarction Registry were analysed retrospectively from November 2005 to December 2007. Prior CVD was defined as having had one or more events of ischaemic or haemorrhagic stroke or a transient ischaemic attack. 3. Of the 12 914 patients reviewed, 906 (7.0%) were found to have had prior CVD. Patients with CVD were older, were more likely to be women and were more likely to have hypertension and diabetes than those without CVD. Patients with prior CVD presented more often with non-ST-segment elevation myocardial infarction and higher Killip class than those without CVD. Furthermore, patients with CVD received less percutaneous coronary intervention (PCI) or thrombolysis compared with those without CVD. Although intensive medical therapy was equal in both groups, clinical outcomes at 8 months showed that patients with CVD had a higher incidence of cardiac death (adjusted odds ratio (OR) 1.42; 95% confidence interval (CI) 1.14-1.76; P = 0.002) and total death (adjusted OR 1.50; 95% CI 1.25-1.81; P < 0.001) than those without CVD. 4. In conclusion, patients with prior CVD presented with worse clinical characteristics on admission and were less likely to receive PCI or thrombolysis than those without CVD. Given the poorer mid-term clinical outcomes, more intensive and aggressive management shouldis recommended for patients with prior CVD to improve their long-term clinical outcome.

Structure-Guided Drug Design of 6-Substituted Adenosine Analogues as Potent Inhibitors of Mycobacterium tuberculosis Adenosine Kinase.

Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 µM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a potentially therapeutically relevant cavity unique to the MtbAdoK homodimer.

A cascade reaction with iminium ion isomerization as the key step leading to tetrahydropyrimido[4,5-d]pyrimidines.

A novel cascade reaction of aminopyrimidines 1 with N-alkyl amino acids or analogues was investigated. The keys to this cascade are the isomerization of an iminium ion formed between the aldehyde group in pyrimidine and the secondary amine of an amino acid, and subsequent cyclization to the neighboring amino group. This sequence could be useful in the synthesis of novel tetrahydropyrimido[4,5-d]pyrimidine libraries.

Structure-guided design of AMP mimics that inhibit fructose-1,6-bisphosphatase with high affinity and specificity.

AMP binding sites are commonly used by nature for allosteric regulation of enzymes controlling the production and metabolism of carbohydrates and lipids. Since many of these enzymes represent potential drug targets for metabolic diseases, efforts were initiated to discover AMP mimics that bind to AMP-binding sites with high affinity and high enzyme specificity. Herein we report the structure-guided design of potent fructose 1,6-bisphosphatase (FBPase) inhibitors that interact with the AMP binding site on FBPase despite their structural dissimilarity to AMP. Molecular modeling, free-energy perturbation calculations, X-ray crystallography, and enzyme kinetic data guided our redesign of AMP, which began by replacing the 5'-phosphate with a phosphonic acid attached to C8 of the adenine base via a 3-atom spacer. Additional binding affinity was gained by replacing the ribose with an alkyl group that formed van der Waals interactions with a hydrophobic region within the AMP binding site and by replacing the purine nitrogens N1 and N3 with carbons to minimize desolvation energy expenditures. The resulting benzimidazole phosphonic acid, 16, inhibited human FBPase (IC50 = 90 nM) 11-fold more potently than AMP and exhibited high specificity for the AMP binding site on FBPase. 16 also inhibited FBPase in primary rat hepatocytes and correspondingly resulted in concentration-dependent inhibition of the gluconeogenesis pathway. Accordingly, these results suggest that the AMP site of FBPase may represent a potential drug target for reducing the excessive glucose produced by the gluconeogenesis pathway in patients with type 2 diabetes.

Discovery of potent and specific fructose-1,6-bisphosphatase inhibitors and a series of orally-bioavailable phosphoramidase-sensitive prodrugs for the treatment of type 2 diabetes.

Excessive glucose production by the liver coupled with decreased glucose uptake and metabolism by muscle, fat, and liver results in chronically elevated blood glucose levels in patients with type 2 diabetes. Efforts to treat diabetes by reducing glucose production have largely focused on the gluconeogenesis pathway and rate-limiting enzymes within this pathway such as fructose-1,6-bisphosphatase (FBPase). The first potent FBPase inhibitors were identified using a structure-guided drug design strategy (Erion, M. D.; et al. J. Am. Chem. Soc. 2007, 129, 15480-15490) but proved difficult to deliver orally. Herein, we report the synthesis and characterization of a series of orally bioavailable FBPase inhibitors identified following the combined discoveries of a low molecular weight inhibitor series with increased potency and a phosphonate prodrug class suitable for their oral delivery. The lead inhibitor, 10A, was designed with the aid of X-ray crystallography and molecular modeling to bind to the allosteric AMP binding site of FBPase. High potency (IC50 = 16 nM) and FBPase specificity were achieved by linking a 2-aminothiazole with a phosphonic acid. Free-energy perturbation calculations provided insight into the factors that contributed to the high binding affinity. 10A and standard phosphonate prodrugs of 10A exhibited poor oral bioavailability (0.2-11%). Improved oral bioavailability (22-47%) was achieved using phosphonate diamides that convert to the corresponding phosphonic acid by sequential action of an esterase and a phosphoramidase. Oral administration of the lead prodrug, MB06322 (30, CS-917), to Zucker Diabetic Fatty rats led to dose-dependent inhibition of gluconeogenesis and endogenous glucose production and consequently to significant blood glucose reduction.