[GAA gene variants and genotype-phenotype correlations in patients with glycogen storage disease type â ¡].

Objectives: To explore the GAA varient spectrum and the genotype-phenotype correlations in patients with glycogen storage disease type Ⅱ (Pompe disease, PD), as well as to estimate the disease incidence based on carrier rate of GAA varients in Guangzhou population. Methods: A total of 57 PD cases were retrospectively enrolled at Guangzhou Women and Children's Medical Center from January 1, 2010 to May 31, 2020. All patients presented symptoms before the age of 18 years. Each diagnosis was further confirmed by GAA enzyme activity and GAA variants. The carrier rate of GAA varients was calculated based on variants detected by whole exon sequencing among 2 395 healthy children in Guangzhou. Results: Among the 57 PD patients (including male 26, female 31),twenty-eight patients with infantile onset PD (IOPD) presented with progressive general muscle weakness and cardiomyopathy. The mean ages of symptom onset and diagnosis were (2.5±1.4) and (5.0±3.0) months, respectively. Twenty-six cases died in the first year after birth.Twenty-three patients with late onset PD (LOPD) presented with progressive muscle weakness. Seven of them had respiratory failure at diagnosis. The mean ages of symptom onset and diagnosis were (12.0±5.0) and (17.0±7.5) years, respectively. Six children with atypical IOPD showed motor delay, muscle weakness and cardiomyopathy. Their diagnosis was confirmed at 2.5-7.0 years of age. Among the 57 patients, 47 different variants were identified in the GAA gene. Three variants: c.797C>T, c.1109G>A and c.1757C>T were novel. c.1935C>A (25/114, 21.9%) and c.2238G>C (15/114, 13.2%) were the most common variants, detected in 57.1% of IOPD and 65.2% (15/23) of LOPD patients, respectively. Among the 28 IOPD patients, 26 cases (92.9%) carried at least one missense variant which indicated positive cross-reactive immunologic material (CRIM). The carrier rate of pathogenic variants in GAA gene among healthy children was 24/2 395. The estimated incidence of PD in this population is about 1/40 000. The frequencies of pseudodeficiency variants c.1726G>A and c.2065G>A homozygotes were 26.3% (15/57) and 35.1% (20/57) in PD patients, which were significantly higher than those (1.7% (40/2 395) and 3.9% (94/2 395)) in healthy children (χ²=151.2, 121.9; both P<0.01). Conclusions: PD presents as a spectrum, some as atypical IOPD. The c.1935C>A and c.2238G>C are common variants, correlated with IOPD and LOPD respectively. The c.796C>T and c.1082C>T are usually found in atypical IOPD. The majority of IOPD patients is predicted to be CRIM positive. The estimated incidence of PD is about 1/40 000.

[Correlation analysis of genotypes and the enzymatic activities of glucose-6-phosphate dehydrogenase in neonates in Guangzhou].

Objective: To reveal the relationship between G6PD genotypes and the G6PD enzyme activities in dried blood spots of newborn screening. Methods: Simple random sampling procedure was used in this study. The fluorescence PCR melting curve analysis was performed to classify G6PD gene variants in 635 neonates coming from Guangzhou Newborn Screening Center during October 1 to 20, 2016, including 15 reported variants. Those samples consisted of 377 cases with screening positive results (261 from males and 116 from females) and 258 cases with screening negative results (32 from males and 226 from females). The cut-off value of G6PD was less than 2.6 U/g Hb in dry blood spots. Sanger sequencing for G6PD gene was used in 7 cases with screening negative results under simple random sampling. One-way ANOVA and least significant difference method (LSD) test were performed to compare the difference of G6PD activity among genotypes. Results: The top 6 frequency of G6PD gene variants were c.1388G>A(35.07%), c.1376G>T(32.13%), c.95A>G(12.72%), c.871G>A(8.32%), c.1024C>T(4.08%) and c.392G>T(2.28%), accounting for 94.62% of all variant alleles (580/613). A total of 253 males positive for enzyme activity were detected to have gene mutations. The positive rate of G6PD enzyme activity was 98.06%(253/258). The mean values of G6PD activities for c.1376G>T,c.95A>G and c.1388G>A were 0.85, 1.10 and 1.28 U/g Hb, respectively. There were significant differences among the three groups (F=28.7, P<0.01). A total of 105 females positive for enzyme activity were detected to have gene mutations. The positive rate of G6PD enzyme activity was 90.52%(105/116). The positive rate of G6PD enzyme activity was 26.95% among 256 females with one point mutation while it was 83.72% in females with multi-allele variants. The G6PD activity of heterozygous females was (2.9±0.8) U/g Hb, which was significant higher than that of females with multi-allele variants (1.5±1.0) U/g Hb (t=8.6,P<0.01). Conclusions: G6PD activities in dried blood spots were related to G6PD genotypes in males. They were also associated with the numbers of allele variants in females. Newborn screening for G6PD deficiency can be used to detect most of G6PD-deficient hemizygotes and female patients with multi-allele variants, which is helpful for preventing neonatal jaundice and medicine application.

[Newborn screening for primary carnitine deficiency and variant spectrum of SLC22A5 gene in Guangzhou].

Objective: To evaluate and improve the performance of the newborn screening program for primary carnitine deficiency (PCD) based on tandem mass spectrometry and to investigate the incidence of PCD and molecular characteristics of SLC22A5 gene in Guangzhou. Methods: A total of 200 180 neonates born in Guangzhou from 2015 to 2019 were enrolled into the newborn screening program for PCD by tandem mass spectrometry at Guangzhou Newborn Screening Center. The positive results of screening for PCD was defined as free carnitine (C0) less than 10 µmol/L with decreased acylcarnitine species in dried blood spots of three to seven days after birth. Screen-positive newborns and their mothers were recalled for another blood spot sample. The diagnosis was confirmed based on decreased levels of C0 and acylcarnitine species in recalled blood spots and genetic analysis in SLC22A5 gene sequencing. The utility of using the sum of propionylcarnitine and palmitoylcarnitine (C3+C16) as a biomarker for acylcarnitine species in newborn screening was retrospectively evaluated. The levels of C0 and (C3+C16) at first screening were compared between newborns with PCD and newborns born to mothers with PCD by independent t test. The variant spectrum and known pathogenic variants carrier rate of SLC22A5 in 2 395 healthy children in Guangzhou Women and Children's Medical Center through whole exon sequencing were analyzed. Results: Among 200 180 neonates, 239 (0.12%) cases were screen-positive for PCD. A total of 37 patients including 15 newborns and 22 mothers had confirmed PCD. The incidence of PCD was 1/13 345 in newborns and 1/9 099 in mothers, respectively. The positive predictive value of this program was 15.5%. Taking cutoff values of C0<8.5 µmol/L or C0 8.5~9.9 µmol/L with (C3+C16)<2 µmol/L, the number of screen-positive cases would be reduced from 810 to 224 without additional false negative case, when compared with cutoff value C0<10 µmol/L only. Both levels of C0 and (C3+C16) at first screening were not significant difference between newborns with PCD and newborns born to mothers with PCD ((6.2±2.4) vs. (5.0±1.8) µmol/L, (1.4±0.4) vs. (1.2±0.5) µmol/L, t=3.826, 0.326; P=0.058, 0.572). Seven PCD mothers experienced moderate fatigue and dizziness in the morning. One of them presented with cardiomyopathy in pregnancy. Genetic analysis of the SLC22A5 gene showed that p.S467C, p.F17L, p.R254X were the three most common variants in newborns with PCD. In PCD mothers and healthy children, the p.S467C, p.F17L and R399W were the three most common whereas the severe variant p.R254X was rare. The population carrier rate for pathogenic variants was 1 in 65 and the estimated incidence of PCD was about 1/16 500. Conclusions: Newborn screening can detect PCD both in newborns and mothers. Adding a quantitative biomarker (C3+C16) <2 µmol/L into the newborn screening program can improve the PCD screen performance. The severe variant p.R253X was common in PCD newborns but rare in PCD mothers and healthy children, indicating that the current screening program maybe failed to detect all PCD newborns and under-estimated the incidence rate of PCD in Guangzhou.

[Newborn screening program and blood amino acid profiling in early neonates with citrin deficiency].

Objective: To investigate the profiles of blood amino acid and acylcarnitine in early neonates with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and the sensitivity of newborn screening, and to explore potential biochemical metabolic markers for newborn screening program. Methods: Amino acid and acylcarnitine profiles in dried blood spots of newborn screening program were analyzed by tandem mass spectrometry (MS/MS). A total of 158 651 neonates born in Guangzhou from January 1, 2015 to June 30, 2019 were enrolled in this newborn screening program, and additionally 55 patients with NICCD confirmed by SLC25A13 gene analysis in Guangzhou Women and Children Medical Center were included in this study. NICCD screen-positive was defined as the cutoff value of citrulline (Cit) ≥ 30 µmol/L. The values of blood sampling time of the true positive group and those of the false negative group were compared by t-test. The levels of amino acid and acylcarnitine among different groups, including true positive group (Cit≥30 µmol/L), false negative group (Cit 21-<30 µmol/L and Cit<21 µmol/L) and the normal control group, were analyzed by F test, respectively. Results: Among 158 651 neonates, 39 neonates were positive for NICCD screening. Three of them were confirmed NICCD and 4 cases were found to be false negatives. The positive predictive value was 7.7% and the sensitivity was about 43.0%. Among 55 patients with NICCD, 18 cases (18/55, 32.7%) were true positives and 37 cases (37/55, 67.3%) were false negatives based on the cutoff value of citrulline in the dried blood spots for newborn screening. The blood sampling time was significantly different between true positive group and false negative group ((4.28±1.6) vs. (2.98±0.74) d, t=4.06, P<0.01). The increased levels of tyrosine((176.0±98.4) µmol/L), methionine ((37.0±26.9) µmol/L) and phenylalanine ((133.0±80.9)µmol/L) in Cit≥30 µmol/L group (n=18) were significantly different as compared with those in the other three groups, respectively (F=117.0, 58.5, 135.0, P<0.01). The levels of arginine ( (10.0±9.2) , (11.0±9.3) , (9.0±17.8) µmol/L), valine ( (119.0±29.8) , (107.6±14.1) , (102±68) µmol/L) and leucine ( (167.0±37.1) , (161.0±37.7) , (163.5±180.6) µmol/L) were not statistically significant among groups of Cit≥30 µmol/L(n=18), Cit21-<30 µmol/L(n=7) and Cit<21µmol/L(n=30,P>0.05), but they were significantly higher than those of the normal control group ((4±3), (78±21), (114.0±31.5) µmol/L, n=1 000), respectively(F=30.1, 23.0, 29.8, P<0.01). Alanine (Ala) ( (150±50) , (156.0±30.2), (168±105), (152±52) µmol/L) levels showed no significant difference (F=0.86, P>0.05) but the ratios of Ala/Cit (1.52±1.44, 6.82±1.56, 12.06±7.71, 19.42±6.27) decreased significantly among the four groups (F=69.0, P<0.05). The acylcarnitine levels showed no statistically significant results among the different groups (P>0.05). With Cit≥30 µmol/L and Ala/Cit<7.5 as cutoff values, the number of screen-positive cases reduced from 39 to 22 cases with no additional false negative case. With Cit≥21 µmol/L and Ala/Cit<7.5 as cutoff values the number of screen-positive cases increased to 117 cases with 1 additional true positive. Conclusions: The profiles of blood amino acid in early neonates with NICCD present the increased levels of multiple amino acids including citrulline, tyrosine, methionine and phenylalanine, and decreased ratio of Ala/Cit. Taking citrulline and ratio of Ala/Cit as screening markers can improve the positive predictive value appropriately. The limited sensitivity of NICCD newborn screening may be related to early blood sampling time.

[Evaluations of newborn screening program performance and enzymatic diagnosis of glucose-6-phosphate dehydrogenase deficiency in Guangzhou].

Objective: To reveal the molecular epidemiologic characteristics of glucose-6-phosphate dehydrogenase (G6PD) gene and to evaluate based on the genetic analysis the newborn screening program performance and enzymatic diagnosis of G6PD deficiency in Guangzhou. Methods: G6PD enzyme activities were measured by quantitative fluorescence assay in dry blood spots of 16 319 newborns(8 725 males, 7 594 females) 3-7 days after birth in Guangzhou Newborn Center. They were born in Guangzhou form Oct. 1 to 20, 2016. The cutoff value of G6PD was less than 2.6 U/g Hb in dry blood spots. G6PD deficiency was diagnosed when G6PD<1 700 U/L or G6PD/6PGD<1 in red blood cells. Genetic analysis of G6PD gene was performed on the dry blood spot samples of 823 newborns (including positive 346, negative 477)with various levels of G6PD enzyme activities through fluorescence PCR melting curve analysis(FMCA) to detect 15 kinds of mutations reported to be common among Chinese.G6PD gene Sanger sequency was performed in seven highly suspicious patients with negative results by FMCA. Results: (1) Using the cutoff value of G6PD< 2.6 U/g Hb , a total of 687(4.2%) newborns showed positive screening results, including 560 (6.4%) males and 127(1.7%) females. (2) Among the newborns with positive screening results, 214 males and 122 females were randomly chosen for G6PD gene analysis. The results showed that 197 (92.1%) males were hemizygote and 108(88.6%) females were mutation carriers with one to four alleles. Among the newborns with negative screening results, 41 males with G6PD 2.6-2.8 U/g Hb and 436 females with G6PD 2.6-4.5 U/g Hb were chosen for genetic analysis.Mutations were detected in 5(12.2%)boys, and 226(51.8%) girls were carriers.G6PD gene Sanger sequency of seven highly suspicious patients showed that c.406C>T, c.551C>T, c.835A>T hemizygote were found in 3 male's samples, respectively. (3) The estimated prevalence of harboring mutation was 6.0% in males and 13.5% in females according to rates of mutation in samples with various levels of G6PD enzyme activities. Six common mutations were c.1388G>A、c.1376G>T, c.95A> G, c.871G>A, c.1024C>T, c.392G>T, accounting for 95.5% of detected alleles .(4) based on results of G6PD gene analysis, the newborn scereening of G6PD deficiency with cutoff value G6PD<2.6 U/g Hb yielded a positive predict value(PPV) of 93.5%, a false-positive rate of 0.5%, and a sensitivity of 99.0% for males. A PPV of 88.5%, a false-positive rate of 0.2% . The prevalence of severe type G6PD deficiency in females was about 1.5%. Compared with to genetic analysis, the sensitivity and PPV of G6PD activity assay in red blood cells were 95.5%, 97.2%, respectively. Conclusions: The prevalence of G6PD deficiency in males was 6.0% in Guangzhou. Six mutations c.1388G>A, c.1376G>T, c.95A>G, c.871G>A, c.1024C>T, c.392G>T accounted for 95.5%. The cutoff value of G6PD<2.6 U/g Hb innewborn screening program and the criteria of biochemical diagnosis could accurately identify G6PD deficiency . Combined with biochemical and molecular analysis will improve the accuracy of diagnosis of G6PD deficiency and detect more heterozygous females.

[Genetic analysis of TPO, DUOX2 and DUOXA2 genes in children with permanent congenital hypothyroidism suspected dyshormonogenesis].

Objective: To explore the TPO, DUOX2 and DUOXA2 genotypes and phenotypes of children with permanent congenital hypothyroidism(PCH) suspected dyshormonogenesis in Guangzhou, identified and treated at Guangzhou Newborn Screening Center. Six of them were born between 2011 and 2012. Method: Retrospectively analyzed the clinical data of 9 children with PCH suspected dyshormonogenesis. Genetic analysis of TPO, DUOX2 and DUOXA2 genes were performed with Sanger sequencing. Result: Of the 9 patients, four were identified variants in TPO gene including three cases with biallelic variants and one case with monoallelic variant. Novel c. 1784G>C( p. R595T) variant in TPO was predicted to be damaging by SIFT and PolyPhen-2. Four patients harbored monoallelic known variants in DUOX2 gene and the other one harbored heterozygous known mutation c. 738C>G(p.Y246X) in DUOXA2 gene.Two adolescent patients with biallelic variants in TPO gene showed classical PCH phenotypes with thyroid goiter or nodules. The six patients with monoallelic variant in TPO, DUOX2 or DUOXA2 presented variable phenotypes. Among the 433 578 newborns in the 2011-2012 cohort, there were 156 cases of CH. Six of these cases were PCH suspected dyshormonogenesis, among which 1 case was confirmed TPO biallelic variants and 5 cases were monoallelic variants of TPO, DUOX2, or DUOXA2 genes. Conclusion: TPO and DUOX2 variants are the common molecular pathogenesis in children with PCH suspected dyshormonogenesis. Monoallelic variants in TPO, DUOX2 or DUOXA2 are associated with PCH and showed wide variability in their phenotypes. The novel variant p. R595T in TPO is probably a pathologic variant. The prevalence of PCH caused by TPO gene defects is rare in Guangzhou.

[Effect of magnesium and selenium on the expression of matrix metalloproteinases-20 and kallikrein 4 in fluorosis mice].

OBJECTIVE: To study the effect of magnesium/selenium on the expression of matrix metalloproteinases-20(MMP-20) and kallikrein 4(KLK4) during fluorosis in mice and to explore the formation mechanism of dental fluorosis. METHODS: Eighty SPF male ICR mice were randomly divided into 8 groups according to body weight: control group, magnesium group, selenium group, magnesium-selenium group, fluoride group, magnesium-fluorine group, selenium-fluorine group and magnesium-selenium-fluorine group. Mice in control, magnesium, selenium and magnesium-selenium groups were fed double steamed water, and mice in the other four groups were feddouble steamed water with 50 mg/L F(-). Mice in control and fluoride groups were fed conventionally. Mice in magnesium and magnesium-fluorine groups were fed conventionally by adding MgSO4·7H2O 162.5 mg/kg. Mice in selenium and selenium-fluorine groups were fed conventionally by adding Na2SeO3·5H2O 2 mg/kg. Mice in magnesium-selenium and magnesium-selenium-fluorine groups were fed conventionally by adding MgSO4·7 H2O 162.5 mg/kg + Na2SeO3·5H2O 2 mg/kg. Incisor specimens were obtained after the mice were put into death when they were 42 days. The expressions of MMP-20 and KLK4 were observed by using immunohisto-chemicalstain. RESULTS: The meangray value of MMP-20 of fluoride group(133.1±10.3) was significantly higher than that of control group(116.8±10.0), magnesium group (113.6 ± 9.6), magnesium-selenium group(108.2 ± 15.2), magnesium-fluorine group(111.1 ± 8.1) and magnesium-selenium-fluorine group(108.2 ± 11.0), respectively(F=3.864, P<0.05). The mean gray value of MMP-20 of magnesium-selenium-fluorine group(108.2±11.0) was significantly lower than that of selenium group(125.4 ± 7.9), fluoride group (133.1 ± 10.3) and selenium-fluorine group(126.2 ± 2.8), respectively(F= 3.864, P<0.05). The mean gray value of KLK4 of magnesium-selenium group(117.2±11.7) was significantly lower than others(137.3±7.9 of control group, 144.2±7.7 of magnesium group, 138.9±13.3 of selenium group, 149.7 ± 12.4 of fluoride group, 148.9 ± 7.5 of magnesium-fluorine group, 140.6 ± 17.0 of selenium-fluorine group and 140.7 ± 7.3 of magnesium-selenium-fluorine group, F=3.668, P<0.05). In factorial analysis of fluorosis mice, magnesium had effect on the expression of MMP-20(F=42.613, P<0.05), selenium had effect on the expression of KLK4(F=6.649, P<0.05). CONCLUSIONS: The excessive fluoride could inhibit the expressions of MMP-20. The excessive fluoride hadno significant influence on the expression of KLK4. Magnesium and selenium had antagonistic effect on the dental fluorosis.

Increased risk of brain tumor in patients with Parkinson's disease: a nationwide cohort study in Taiwan.

OBJECTIVES: Parkinson's disease (PD) is a neurodegenerative disease. A decreased risk of cancer, except for melanoma, has been observed in patients with PD. The aim of this study was to evaluate the association between brain tumor and PD in a Taiwanese population. MATERIALS AND METHODS: We used data from the National Health Insurance program of Taiwan. The PD cohort contained 2998 patients, and each patient was frequency-matched, based on age and sex, with 4 people without PD, who were randomly selected from the general population. Cox's proportional hazard regression analysis was conducted to estimate the effects of PD on the risk of brain tumor. RESULTS: The risk of developing brain tumor was significantly higher in patients with PD than in those without PD (adjusted hazard ratio = 2.11; 95% confidence interval (CI) = 1.24-3.59), and benign brain tumor exhibited a particularly elevated risk of 2.16-fold (95% CI = 1.26-3.68). The hazard ratio (HR) for developing a benign brain tumor was higher in female patients with PD than in female patients without PD, with the risk being 2.65-fold (95% CI = 1.30-5.43). An analysis of the two age groups, 50-64 years and ≥65 years, showed that the HR of only the 50-64-year group was significantly higher between the PD and non-PD groups (HR = 2.77, 95% CI = 1.07-7.14). CONCLUSION: The present study showed that Taiwanese patients with PD are at a higher risk of developing brain tumor than the general population. The exact underlying etiologies require further investigation.

Comparison of the photosynthetic characteristics of four Lycoris species with leaf appearing in autumn under field conditions.

The diurnal trends of gas exchange and chlorophyll fluorescence parameters in four Lycoris species (L. houdyshelii, L. aurea, L. radiata var. pumila and L. albiflora) were determined and compared with a portable photosynthesis analysis system. Our study revealed that L. houdyshelii had the lowest light compensation point (LCP), while the other three species had higher LCP (12.37-14.99 µmol m-2 s-1); L. aurea had the highest light saturation point (LSP) (1,189 µmol m-2 s-1), and L. houdyshelii and L. albiflora had lower LSP with the values being 322 and 345 µmol m-2 s-1, respectively, and L. radiata var. pumila showed the intermediate LSP. Both the species L. houdyshelii and L. albiflora exhibited a typical and obvious decline in net photosynthetic rate (P N) during midday, which was not observed in L. aurea. This indicated a possible photoinhibition in L. houdyshelii and L. albiflora as the ratio of variable to maximum fluorescence (Fv/Fm) values were higher in these two species. The minimal fluorescence (F0) values were lower in L. aurea and L. radiata var. pumila. The diurnal changes of transpiration rate (E) in all four species presented only one peak, appearing between 11:00 h or 13:00 h. By using simple correlation analyses, it was observed that the environmental factors affecting P N were different among four species and the main factors were photosynthetic photon flux density (PPFD) and relative humidity especially for L. aurea and L. radiata. The results of studying indicated that the four species could be divided into two groups. The species L. radiata var. pumila and L. aurea were more adapted to a relatively high irradiance, and L. houdyshelii and L. albiflora could be grown in moderate-shade environment in order to scale up their growth and productivity.

Effects of exogenous spermidine on subcellular distribution and chemical forms of cadmium in Typha latifolia L. under cadmium stress.

The effects of foliar spraying with spermidine (Spd) on the subcellular distribution and chemical speciation of Cd in Typha latifolia L. in hydroponic system were investigated. Increased Cd concentration in the medium caused an increase of Cd concentration in all tissues and all subcellular fractions, with the highest Cd level occurring in roots, followed by rhizomes and leaves and the greatest accumulation was in cell walls, soluble fraction and organelle containing fractions ranked next, respectively. The greatest amount of Cd was found in the extraction of 1 mol.L(-1) NaCl, followed by 2% HAC, deionized water, 80% ethanol, residues and the least in 0.6 mol.L(-1) HCl. The subcellular distribution and different chemical forms of Cd both decreased after Spd application and were more inhibited by 0.25 than 0.50 mmol.L(-1) Spd, especially in roots at 0.10 mmol.L(-1) Cd treatment. It could be suggested that the retention of Cd in roots, cell walls binding with metal, metal ion compartmentation in vacuoles and Cd forms existing in undissolved and integrated with pectates and protein play an important role in Cd detoxification and Spd could enhance the tolerance of cattail to Cd stress.

Non-linear optical properties of zinc oxide nanowires.

High quality zinc oxide (ZnO) nanowires were grown on n-type Si (100) using vapor-liquid-solid process. We obtained the photoluminescence spectra of ZnO nanowires based on nonlinear optical process using an ultrashort wavelength femtosecond laser as a pumping source. The spectra shows the second harmonic generation phenomenon, as well as the exciton-exciton collision peak at 388 nm and the green emission peak at 515 nm caused by oxygen vacancy. A laser emission peak near 392 nm was observed when pump intensity surpassed 52 mJ/cm2 and a sharp peak about 0.5 nm wide emerged when the energy intensity reached 700 mJ/cm2. We attribute this excitation process to a two-photon absorption process enhanced by Rabi oscillation.

Synthesis and antitumor properties of N1-acyloxymethyl derivatives of bis(2,6-dioxopiperazines).

Many N1-acyloxymethyl derivatives VI of bis(2,6-dioxopiperazine) I, ICRF-154, were prepared and tested for antitumor activity. The treatment of I with formaldehyde gave a crystalline bis(N1-hydroxymethyl) derivative VII, which was acylated under various conditions to give bis(N1-acyloxymethyl) derivatives VI. Antitumor activity of VI against P388 leukemia in mice was studied. Several bis(N1-acyloxymethyl) compounds such as phenylacetyloxymethyl VI-6, methoxycarbonyloxymethyl VI-41, isobutoxycarbonyloxymethyl VI-44, and furancarboxymethyl VI-38 compounds were found to have potent antitumor activities. On the other hand, water-soluble esters having an amine or a carboxylic acid function in their acyl groups showed rather reduced activity. These bis(N1-acyloxymethyl) derivatives VI were presumably hydrolyzed into the parent bis(2,6-dioxopiperazine) I by nonspecific esterase in the body to exhibit their antitumor activity.