[Microbiota structure and diversity in Aedes albopictus at different developmental stages].

OBJECTIVE: To investigate the diversity and composition of microflora in laboratory-reared Aedes albopictus at different developmental stages and larval habitat waters. METHODS: The larval habitat waters and different developmental stages of laboratory-reared A. albopictus were collected, and the V3 and V4 regions of the bacterial 16S rRNA gene were sequenced using Illumina Miseq next-generation sequencing. The abundance and diversity of the microflora were examined using alpha diversity index in A. albopictus at different developmental stages, and the difference in the microflora compositions was compared in A. albopictus at different developmental stages using principal component analysis (PCA). In addition, the species composition and relative abundance of microflora in A. albopictus at different developmental stages were described using histograms and Venn diagrams. RESULTS: A total of 16 phyla, 30 classes, 72 orders, 129 families and 224 genera of bacteria were detected in larval habitat waters and different developmental stages of A. albopictus. The highest bacterial diversity was seen in larval A. albopictus, with Chao index of 125.20 ± 30.48 and Shannon diversity index of 2.04 ± 0.39, which were comparable to those (Chao index of 118.52 ± 15.07 and Shannon diversity index of 2.15 ± 0.30) in larval habitat waters (t = 0.35 and -0.41, both P values > 0.05). The bacterial abundance and evenness were significantly greater in female adults than in male adults (Chao index: 42.50 ± 3.54 vs. 18.50 ± 2.13, t = 8.23, P < 0.05; Shannon diversity index: 1.25 ± 1.67 vs. 0.50 ± 0.05, t = 6.00, P < 0.05). Proteobacteria, Bacteroidota, Actinobacteriota and Finnicutes were four common phyla of bacteria at each developmental stage of A. albopictus, with Proteobacteria dominated at the pupal stage (90.36%), Bacteroidota dominated at the adult stage (46.01% in female adults and 86.11% in male adults), and Actinobacteriota dominated at the larval stage (32.10%). Elizabethkingia and Rahnella 1 were common dominant genera of bacteria at each developmental stage of A. albopictus, with Rahnellal as the major component at the pupal stage (87.56%), Elizabethkingia as the main component at the adult stage (46.01% in female adults and 86.11% in male adults, respectively), and Microbacteria as the dominant bacterial genus at the larval stage (12.11%). In addition, Delftia, Elizabethkingia, Romboutsia, Serratia, Rahnella 1, Enterococcus and Microbacterium were common genera of bacteria at each developmental stage of A. albopictus, with Edaphobaculum dominated at the larval stage (17.54%) and Sphingobacterium dominated in larval habitat waters (13.93%). CONCLUSIONS: There are differences in the composition of symbiotic bacteria at different developmental stages of A. albopictus; however, similar microflora diversity is maintained at the phylum level. The microflora diversity is comparable in larvae and larval habitat waters of A. albopictus.

[Harder maintained than achieved: Challenges of maintaining malaria-free in China].

On June 2021, China was certified malaria-free by WHO. However, the global number and death of malaria cases have recently increased, and the malaria vectors will continue to inhabit in China, resulting in a high difficulty in consolidation of malaria elimination achievements. Hereby, we analyze the current challenges and propose the future priority of the national malaria control program in China, in order to provide insights into prevention of re-establishment of imported malaria in the country.

[Time to initial diagnosis of imported malaria and its influencing factors in Jiangsu Province].

OBJECTIVE: To investigate the health-seeking behaviors of imported malaria cases after returning to China, and to investigate the factors affecting the time to initial diagnosis, so as to provide the scientific evidence for early identification of imported malaria cases and prevention of severe cases development and secondary transmission. METHODS: The individual demographic features, and the disease onset and the time to initial diagnosis of imported malaria cases in Jiangsu Province in 2019 were captured from the National Notifiable Disease Report System and the Information Management System for Parasitic Disease Control in China. The characteristics of health-seeking behaviors and epidemiological features of imported malaria cases were descriptively analyzed, and the factors affecting the time to initial diagnosis of imported malaria cases after returning to China were identified using multivariate logistic regression analysis. RESULTS: A total of 244 imported malaria cases were reported in Jiangsu Province in 2019, and the time to initial diagnosis of the cases were 1-12 days, with mean time of (1.53 ± 1.65) days, with median time of one day. The highest number of malaria cases seeking healthcare services were found on the day of developing primary symptoms (76 cases, 31.1%), followed by on the second day (68 cases, 27.9%), on the third day (46 cases, 18.9%), and 54 cases (22.1%) received initial diagnosis 3 days following presence of primary symptoms, including 3 cases with initial diagnosis at more than one week. High proportions of imported malaria cases with a delay in the time to initial diagnosis were seen in migrant workers who returned to China in January (14 cases, 5.7%) and December (13 cases, 5.3%) and those aged between 41 and 50 years (32 cases, 13.1%). Multivariate logistic regression analysis showed relative short time to initial diagnosis among imported malaria cases returning to China on March [odds ratio (OR) = 0.16, P = 0.03, 95% confidence interval (CI): (0.03, 0.85)] and those with a history of overseas malaria parasite infections [OR = 0.36, P = 0.001, 95% CI: (0.19, 0.67)]. CONCLUSIONS: Timely health-seeking behaviors should be improved among imported malaria cases in Jiangsu Province, patients with a history of overseas malaria infections require faster health-seeking activities.

[Surveillance of Culex mosquitoes in Jiangsu Province from 2018 to 2019].

OBJECTIVE: To investigate the distribution and density of Culex mosquito populations and the resistance of Culex pipiens pallens to insecticides in Jiangsu Province in 2018 and 2019. METHODS: During the period from June to October in 2018 and 2019, six counties (districts, cities) were sampled in southern, northern and central Jiangsu Province as surveillance sites. The density of Culex mosquitoes was measured overnight using the light trapping technique. In addition, Culex pipiens pallens mosquitoes were collected from Hai'an of Nantong City and Yandu District of Yancheng City, central Jiangsu Province, and the sensitivity of female first filial generations to dichlorodiphenyltrichloroethane (DDT), malation, proposur, beta cypermethrin and deltamethrin was tested using the standard WHO insecticide susceptibility test assay. RESULTS: A total of 104 423 Culex mosquitoes were captured in six surveillance sites of Jiangsu Province in 2018 and 2019, and Culex quinquefasciatus (49.11%), Culex pipiens pallens (28.38%), and Culex tritaeniorhynchus (21.04%) were predominant species. The density of Culex mosquitoes started to increase since early June, peaked in July and tended to be low in late October. Culex pipiens pallens mosquitoes captured from Hai'an was susceptible to malation, while those from Yandu District were moderately resistant to malation. Culex pipiens pallens mosquitoes from both Yandu and Hai'an were moderately resistant to proposur, and were resistant to DDT, beta cypermethrin and deltamethrin. CONCLUSIONS: Culex quinquefasciatus, Culex pipiens pallens and Culex tritaeniorhynchus are predominant Culex species in Jiangsu Province. Culex pipiens pallens is resistant to DT, beta cypermethrin and deltamethrin in central Jiangsu Province.

[Study on emergency metabolic changes of Anopheles sinensis larvae following exposure to deltamethrin].

OBJECTIVE: To analyze the changes of small molecular metabolites in the larvae of a deltamethrin-sensitive strain of Anopheles sinensis following exposure to deltamethrin, so as to provide the scientific basis for investigating the metabolic pathway and screening metabolic markers of deltamethrin in An. sinensis. METHODS: The 50% and 75% lethal concentrations (LC50 and LC75) of deltamethrin against the larvae of a deltamethrin-sensitive strain of An. sinensis were calculated in laboratory. The type and content of An. sinensis larvae metabolites were detected using high performance liquid chromatography and mass spectrometry (LC-MS/MS) following exposure to deltamethrin at LC50 and LC75 for 30 min and 24 h, and the changes of metabolites were analyzed. RESULTS: The LC50 and LC75 values of deltamethrin were 4.36 × 10-3 µg/mL and 1.12 × 10-2 µg/mL against thelarvae of a deltamethrin-sensitive strain of An. sinensis. Following exposure of the larvae of a deltamethrin-sensitive strain of An. sinensis to deltamethrin at LC50 and LC75 for 30 min, the differential metabolites mainly included organic oxygen compounds, carboxylic acid and its derivatives, fatty acyl and pyrimidine nucleotides, with reduced glucose levels. Following exposure for 24 h, the differential metabolites mainly included organic oxygen compounds, carboxylic acid and its derivatives, aliphatic acyl and purine nucleotides, with increased glucose level detected. CONCLUSIONS: Carbohydrate, carboxylic acid and its derivatives, fatty acyls, amino acids and their derivatives may play important roles in deltamethrin metabolism in the larvae of a deltamethrin-sensitive strain of An. sinensis.

[Challenges and countermeasures in prevention of re - establishment of imported malaria in China].

Malaria was one for the most serious communicable diseases in China. Following the concerted efforts for decades, remarkable achievements have been gained for malaria control in China. Since the national malaria elimination programme was initiated in China in 2010, local malaria transmission was rapidly interrupted, with zero indigenous malaria case reported for the first time in the country in 2017, and the country will undergo the certification of malaria elimination by WHO. Currently, however, malaria remains hyper-endemic across the world. In China, there are more than 2 000 overseas imported malaria cases each year, and prevention of re-establishment of imported malaria will become the major task in future malaria control activities. Here by, we analyze the main challenges in the prevention of re-establishment of imported malaria in China, and propose the corresponding countermeasures, so as to provide insights into the consolidation of malaria elimination achievements.

[Epidemiological characteristics of imported Plasmodium ovale malaria in Jiangsu Province from 2012 to 2020].

OBJECTIVE: To analyze the epidemiological characteristics of imported cases with Plasmodium ovale infections in Jiangsu Province from 2012 to 2020, so as to provide insights into the development of the imported malaria control strategy in the province. METHODS: All data pertaining to cases with definitive diagnosis of P. ovale malaria in Jiangsu Province from 2012 to 2020 were captured from the National Notifiable Disease Report System and the Information Management System for Parasitic Disease Control in China, including the date of going abroad and returning to China, time of malaria infections overseas, date of malaria onset, initial diagnosis and definitive diagnosis. All data pertaining to epidemic status were descriptively analyzed. RESULTS: A total of 347 cases of P. ovale malaria were reported in Jiangsu Province from 2012 to 2020, with the highest number seen in 2015 (71 cases). All cases were laboratory-confirmed overseas imported malaria cases, accounting for 14.32% of all reported malaria cases in Jiangsu Province during the period from 2012 to 2020. The 5 cities with the highest number of imported P. ovale malaria cases included Lianyungang City (53 cases, 15.27%), Nantong City (44 cases, 12.68%), Huai'an (44 cases, 12.68%), Taizhou City (44 cases, 12.68%) and Yangzhou City (36 cases, 10.37%). The highest number of imported P. ovale malaria cases was reported in October (39 cases, 11.24%), and the lowest number was seen in December (21 cases, 6.05%). P. ovale infections mainly occurred in were Equatorial Guinea (97 cases, 37.95%), Angola (60 cases, 17.29%) and Nigeria (40 cases, 11.53%). The median duration between returning to China and malaria onset was 64 (144) days, and 7.49% (26/347) of all cases developed malaria one year after returning to China. The initial diagnosis of P. ovale malaria was mainly made at county-level medical institutions (117 cases, 33.72%), and the definitive diagnosis was mainly made at city-level medical institutions (122 cases, 35.16%). The correct rate of initial diagnosis of P. ovale malaria increased from 0 in 2012 to 78.26% in 2020, appearing a tendency towards a rise year by year (χ2 = 50.90, P < 0.01). CONCLUSIONS: Imported P. ovale malaria cases were reported in Jiangsu Province each year from 2012 to 2020, and P. ovale infections predominantly occurred in Africa. Initial and definitive diagnoses of P. ovale malaria were mainly made at city- and county-level medical institutions. Training on the detection ability of malaria parasites is recommended among grassroots microscopists to improve the diagnostic ability of P. ovale malaria, and consolidate the achievements of malaria elimination in Jiangsu Province.

[Strategies and studies of malaria vector control in Jiangsu Province].

Malaria was one of the major infectious diseases in Jiangsu Province, where Anopheles sinensis and An. anthropophagus are main vectors for malaria transmission. Following the concerted efforts for decades, the goal of malaria elimination was achieved in Jiangsu Province in 2019, and the vector control strategy has played a vital role during the progress towards malaria elimination in Jiangsu Province. Hereby, we review the historical distribution and ecological features of An. sinensis and An. anthropophagus and describe vector control strategies at different stages of malaria control in Jiangsu Province. In addition, the advances in the research of vector biology and control in Jiangsu Province are discussed, including vector identification, strain colonization, susceptibility to malaria parasites and insecticide resistance.

[Challenges and countermeasures on Chinese malaria elimination programme during the coronavirus disease 2019 (COVID-19) outbreak].

Since the end of 2019, the coronavirus disease 2019 (COVID-19) has been extensively epidemic in China, which not only seriously threatens the safety and health of Chinese people, but also challenges the management of other infectious diseases. Currently, there are still approximately three thousand malaria cases imported into China every year. If the diagnosis and treatment of malaria cases as well as the investigation and response of the epidemic foci are not carried out timely, it may endanger patients'lives and cause the possible of secondary transmission, which threatens the achievements of malaria elimination in China. Due to the extensive spread and high transmission ability of the COVID-19, there is a possibility of virus infections among malaria cases during the medical care-seeking behaviors and among healthcare professionals during clinical diagnosis and treatment, sample collection and testing and epidemiological surveys. This paper analyzes the challenges of the COVID-19 for Chinese malaria elimination programme, and proposes the countermeasures in response to the COVID-19 outbreak, so as to provide the reference for healthcare professionals.

[Epidemic situation of malaria in Jiangsu Province in 2019].

OBJECTIVE: To analyze the epidemic situation of malaria in Jiangsu Province in 2019, so as to provide the scientific basis for the development of the strategy for the prevention of re-introduction of imported malaria. METHODS: The malaria case report information, epidemiological case investigation information, epidemic foci investigation and management report in Jiangsu Province in 2019 were collected, and all epidemiological data were descriptively analyzed. RESULTS: A total of 244 malaria cases were reported in Jiangsu Province in 2019, and all cases were laboratory-confirmed overseas imported cases, including 4 cases with vivax malaria, 206 cases with falciparum malaria, 12 cases with malariae malaria and 22 cases with ovale malaria. In 2019, there were 12 malaria cases progressing into severe cases in Jiangsu Province, with one death. Nanjing, Nantong, Lianyungang, Taizhou and Changzhou cities contributed the largest number of malaria cases in 2019, with the number of malaria cases accounting for 59.84% of total cases in Jiangsu Province. The infections occurred in Papua New Guinea (2 cases), Pakistan (1 case) and 27 African countries (241 cases), including Angola, the Democratic Republic of the Congo, Nigeria, Equatorial Guinea, Cote d'Ivoire and so on. There were 77 cases (31.55%) with a visit to doctor on the day of onset, and 146 cases (59.84%) within 1 to 3 days after onset. In addition, there were 149 cases (61.06%) with definitive diagnosis at the first visit and 77 cases (31.55%) diagnosed within 1 to 3 days after the visit, and the mean duration from the visit to definitive diagnosis was (0.80 ± 1.59) d, which significantly shortened as compared to that (1.34 d ± 2.59 d) in 2018 (U = 2.53, P < 0.05). CONCLUSIONS: Intensifying the surveillance and management of imported malaria and improving the diagnostic capability of imported malaria and the treatment of severe malaria cases are required to consolidate the achievements of malaria elimination in Jiangsu Province.

[Epidemic situation of malaria in Jiangsu Province in 2018].

OBJECTIVE: To analyze the epidemic situation of malaria in Jiangsu Province in 2018, so as to provide scientific evidence for formulating post-elimination malaria surveillance schemes and technical measures in Jiangsu Province. METHODS: The malaria case report cards, epidemiological individual investigation forms of malaria cases and foci data were collected from Jiangsu Province in 2018, and the epidemic situation of malaria was descriptively analyzed. RESULTS: A total of 243 malaria cases were reported in Jiangsu Province in 2018, which increased by 1.67% in relative to in 2017 (239 cases), and these cases included 171 cases with falciparum malaria, 14 cases with vivax malaria, 15 cases with quartan malaria, 42 cases with ovale malaria and a case with mixed infection of P. vivax and P. ovale. All cases were overseas imported, and no local secondary cases were found. The malaria cases were predominantly workers (76.54%). Nantong City (48 cases), Yangzhou City (33 cases) and Taizhou City (22 cases) were the most 3 cities with the largest number of malaria cases across Jiangsu Province. The malaria infections predominantly occurred in African areas (96.30%), and the other 9 cases had infections in Asia (8 cases) and Central America (1 case). There were 125 cases (51.44%) and 91 cases (37.45%) with definitive diagnosis at the day of admission and within 1 to 3 days post-admission, respectively. The percentages of definitive diagnosis at initial diagnosis were 48.27%, 88.76% and 97.30% at township-, county- and city-level medical institutions, respectively, and the percentage of definitive diagnosis at initial diagnosis was significantly lower in township-level medical institutions than in county- (χ2 = 21.47, P < 0.01) and city-level medical institutions (χ2 = 32.86, P < 0.01). CONCLUSIONS: There are no local malaria cases in Jiangsu Province; however, the number of overseas imported malaria cases remains high in China. In the future, improving the post-elimination malaria surveillance system, enhancing the awareness of malaria prevention and control knowledge among high-risk populations, increasing the diagnostic capability of malaria in medical institutions, and improving the management of imported malaria cases should be performed to consolidate the achievements of malaria elimination.

[Current status of malaria rapid diagnostic testing strips application in primary-level healthcare providers in Jiangsu Province].

Objective: To investigate the current status of malaria rapid diagnostic test (RDT) strips application and malaria laboratory technicians' evaluation about them at primary healthcare provider level in Jiangsu Province. Methods: From November to December 2016, 878 medical institutions and 118 CDCs of city, county and township/community level in Jiangsu Province were selected as study samples using stratified random sampling method. Self-designed questionnaire was distributed to investigate the institution's malaria work task, RDT strips application and evaluation status in 2015. We also investigated the socio-demographic information and collected the RDT strips evaluation score from the malaria laboratory technicians selected from the institutions investigated (one technician from each institution). Rank sum test was performed to compare the RDT strips evaluation scores between medical institutions and CDCs, and among different medical institutions and CDCs. Results: In 2015, 405 cases of malaria were reported, 362 200 person-time of malaria blood testing task was conducted, and 100 000 RDT strips were procured and provided for healthcare providers in Jiangsu province for free. Of the 996 healthcare institutions investigated, 628 used RDT strips in the year 2015 and the median (P(25), P(75)) of RDT strips volume used in these institutions was 10 (2, 25). The volume of RDT strips used in CDCs (15 (5, 52)) was significantly higher than that in medical institutions (10 (2, 25), (Z=3.42, P=0.001)). The investigated CDCs gave higher score on RDT strips' testing time per operation (10 (8.5, 10)) than medical institutions (9(8, 10), (Z=-2.20, P=0.028)). The employers of 614 investigated malaria laboratory technicians used RDT strips in 2015. The median of the scores given by CDC malaria laboratory technicians for RDT strips in terms of testing time per operation, testing operation and results judgement difficulties were 10 (9, 10), 10 (9, 10) and 10 (9, 10), respectively, which were significantly higher than those from technicians of medical institutions (9 (8, 10), 9 (8, 10), 9 (8, 10), (Z values were -2.55, -2.97 and -2.96, respectively; P values were all less than 0.05)). Conclusion: RDT strips had been widely performed in health institutions in Jiangsu Province. The amount of RDT strips used in CDCs was significantly higher than that in medical institutions. Primary-level institutions and malaria laboratory technicians generally recognized RDT strips' advantage for application in terms of testing time and operational procedure. CDCs and malaria laboratory technicians from them gave higher regards on RDT strips in terms of testing time per operation, testing operation and results judgement difficulties compared with that of medical institutions.

[Cases diagnosis of imported malaria in Jiangsu province, 2014-2016].

Objective: To understand the situation related to health seeking and diagnosis of imported malaria and to provide practical measures for malaria elimination in Jiangsu province. Methods: Data on imported malaria cases in Jiangsu province was retrieved in CISDCP from 2014 to 2016. Relevant information on health seeking behavior, diagnosis and treatment of the disease was gathered. Results: A total of 1 068 imported cases were reported in Jiangsu province from 2014 to 2016. Except for one malaria case that was caused by blood transfusion, the rest patients were all recognized as 'imported'. Majority of the cases were migrant laborers working in African countries. The accurate rates on the diagnosis of ovale, vivax and quartan malaria and mixed infection were relatively low, as 79.3% (107/135), 29.5% (18/61), 52.9% (18/34) and 0.0% (0/2) at the primary health care settings, respectively. Rate of seeking health care on the same day of onset was more in 2015 than in 2014 and 2016 (χ(2)=18.6, P=0.001). While only 65.4% (699/1 068) of the patients were diagnosed correctly at the primary health care settings. There appeared no statistical difference in the 3-year-study period (χ(2)=5.4, P=0.246). Capacity on 'correct diagnosis' seemed stronger at the CDC than at the hospital levels (χ(2)=13.2, P=0.000; χ(2)=5.4, P=0.020). Totally, 72.7% (32/44) of the severe falciparum malaria cases did not immediately seek for health care when the symptoms started. Conclusions: Migrant workers returning from the high endemic malaria areas seemed to have poor awareness in seeking health care services. Capability on correct diagnosis for malaria at the primary health care settings remained unsatisfactory and staff from these settings needs to receive adequate training.

Selective inhibition of ICAM-1 and E-selectin expression in human endothelial cells. 2. Aryl modifications of 4-(aryloxy)thieno[2,3-c]pyridines with fine-tuning at C-2 carbamides.

The elevated expression of cell adhesion molecules (CAMs) on the lumenal surface of vascular endothelial cells is a critical early event in the complex inflammatory process. The adhesive interactions of these CAMs that include E-selectin, ICAM-1, and VCAM-1 with their counter-receptors on leukocytes, such as integrins of the alpha(L)beta(2) family, result in migration of the leukocytes to the site of inflammation and cause tissue injury. Pharmaceutical agents that could suppress the induced expression of one or more of these cell adhesion molecules would provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A-205804 (1), a potent and selective inhibitor of the induced expression of E-selectin and ICAM-1 over VCAM-1, was further modified with emphasis at the C-4 and C-2 positions to identify a more potent drug candidate with a good pharmacokinetic profile and physical properties. Replacement of the C-4 sulfur linkage in 1 with an oxygen atom eliminated one of the two major metabolites for this lead molecule. The para-position of the 4-phenoxy group of the thieno[2,3-c]pyridine lead is found to be very critical for a higher in vitro potency and selectivity of E-selectin and ICAM-1 over VCAM-1 expression. This position is presumably close to the solvent-accessible region of the target protein-inhibitor complex. An attempt to install a water-solubilizing group at the para-position of the phenoxy group to increase the aqueous solubility of this lead series through various linkages failed to provide an ideal inhibitor. Only small substituents such as fluorine are tolerated at the meta- and ortho-positions of the 4-phenoxy to retain a good in vitro potency. Bromo, trifluoromethyl, pyrazol-1-yl, and imidazol-1-yl are among the better substituents at the para-position. With fine-tuning at the C-2 position we discovered a series of very potent (IC(50) < 5 nM for ICAM-1) and selective (>200-fold vs VCAM-1) inhibitors with a good pharmacokinetic profile. Demonstrated efficacy in a rat rheumatoid arthritis model and in a mice asthma model with selected compounds is also reported.

Discovery of inhibitors of cell adhesion molecule expression in human endothelial cells. 1. Selective inhibition of ICAM-1 and E-selectin expression.

A critical early event in the inflammatory cascade is the induced expression of cell adhesion molecules on the lumenal surface of vascular endothelial cells. These adhesion molecules include E-selectin, ICAM-1, and VCAM-1, which serve to recruit circulating leukocytes to the site of the inflammation. These adhesive interactions allow the leukocytes to firmly adhere to and cross the vascular endothelium and migrate to the site of tissue injury. Pharmaceutical agents which would prevent the induced expression of one or more of the cell adhesion molecules on the endothelium might be expected to provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A thieno[2,3-d]pyrimidine, A-155918, was identified from a whole-cell high-throughput assay for compounds which inhibited the tumor necrosis factor-alpha (TNFalpha)-induced expression of E-selectin, ICAM-1, or VCAM-1 on human vascular endothelial cells. Traditional medicinal chemistry methods were applied to this low-micromolar inhibitor, resulting in the 2,4-disubstituted thieno[2,3-c]pyridine A-205804, a potent and selective lead inhibitor of E-selectin and ICAM-1 expression (IC(50) = 20 and 25 nM, respectively). The relative position of the nitrogen atom in the thienopyridine isomer was shown to be critical for activity, as was a small amide 2-substituent.

Conformationally restricted mimics of vitamin D rotamers.

Drug developments in the vitamin D field have continued to focus on structure-function studies of analogs produced by chemically modifying the structure of 1alpha,25-dihydroxyvitamin D(3) (1,25-D3) and its metabolites. Direct structural information gleaned from X-ray crystallographic or NMR studies regarding the ligand-receptor complex and other guest-host systems, which are likely involved in initiating biologic responses, also offers potential insight into drug design. Evidence has accrued suggesting that topologically different conformers of 1,25-D3 may bind to proteins in different ways, including the induction of different conformations of protein. This paper concerns our progress on the chemical synthesis of analogs (e.g. ansa-steroids, suprasterols, vinylallenes and other analogs) conformationally locked or at least rotationally restricted to mimic higher energy conformers of 1,25-D3.

Biological activity of CD-ring modified 1alpha,25-dihydroxyvitamin D analogues: C-ring and five-membered D-ring analogues.

Nonsteroidal analogues of 1alpha,25(OH)2D3, lacking either the full five-membered D ring (C-ring analogues) or the full six-membered C ring (D-ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1alpha,25(OH)2D3. Maximal superagonistic activity was seen for the C-ring analogue with a 24(R)-hydroxyl group in the side chain [30- to 60-fold the activity of 1alpha,25(OH)2D3]. The 19-nor-16-ene-26,27-bishomo C-ring analogue showed the best ratio of antiproliferative to calcemic effects (1275-fold better than 1alpha,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D-dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR-ligand complex more resistant against protease digestion than is 1alpha,25(OH)2D3. In contrast to 20-epimer steroidal vitamin D analogues, 20-epimer C-ring analogues were less potent than analogues with a natural C-20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1alpha,25(OH)2D3 despite lower receptor affinity and, for the C-ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published.

Synthesis, biological activity, and conformational analysis of four seco-D-15,19-bisnor-1alpha,25-dihydroxyvitamin D analogues, diastereomeric at C17 and C20.

The synthesis of four CD-ring-modified 19-nor-1alpha, 25-dihydroxyvitamin D(3) derivatives lacking C15, referred to as 6C analogues, and diastereomeric at C17 and C20 is described. The synthesis involves an Ireland-Claisen rearrangement of a 3-methyl-substituted ester of (1R)-3-methyl-2-cyclohexen-1-ol as the key step, followed by elaboration of the side chain, transformation into a C8 cyclohexanone derivative, and final Wittig-Horner coupling with the 19-nor A-ring phosphine oxide. Despite possessing a more flexible side chain than the parent hormone, biological evaluation showed an unexpected superagonistic antiproliferative and prodifferentiating activity (10-50 times higher as compared to that of 1alpha,25(OH)(2)D(3)) for the diastereomer with the "natural" configuration at C17 and C20. The other diastereomers exhibit a 25-90% decrease in activity. All four analogues show a decreased binding affinity (45% or less), and their calcemic activity is 4-400 times less than that of 1alpha,25(OH)(2)D(3). The conformational behavior of their side chain was studied using molecular mechanics calculations, and the result is presented as volume maps. A relative activity volume was determined by subtraction of the volume map of the least active analogue from the volume map of the most active one. This shows three regions corresponding to preferred orientations in space of the side chain of the active analogue. One of these regions was found to overlap with the region that is preferentially occupied by the most active of the four diastereomeric 22-methyl-substituted 1alpha,25(OH)(2)D(3) analogues.