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Lithium nickel manganese cobalt oxide (NMC) is a commercially successful Li-ion battery cathode due to its high energy density; however, its delivered capacity must be intentionally limited to achieve capacity retention over extended cycling. To design next-generation NMC batteries with longer life and higher capacity the origins of high potential capacity fade must be understood. Operando hard X-ray characterization techniques are critical for this endeavor as they allow the acquisition of information about the evolution of structure, oxidation state, and coordination environment of NMC as the material (de)lithiates in a functional battery. This perspective outlines recent developments in the elucidation of capacity fade mechanisms in NMC through hard X-ray probes, surface sensitive soft X-ray characterization, and isothermal microcalorimetry. A case study on the effect of charging potential on NMC811 over extended cycling is presented to illustrate the benefits of these approaches. The results showed that charging to 4.7 V leads to higher delivered capacity, but much greater fade as compared to charging to 4.3 V. Operando XRD and SEM results indicated that particle fracture from increased structural distortions at >4.3 V was a contributor to capacity fade. Operando hard XAS revealed significant Ni and Co redox during cycling as well as a Jahn-Teller distortion at the discharged state (Ni 3+ ); however, minimal differences were observed between the cells charged to 4.3 and 4.7 V. Additional XAS analyses using soft X-rays revealed significant surface reconstruction after cycling to 4.7 V, revealing another contribution to fade. Operando isothermal microcalorimetry (IMC) indicated that the high voltage charge to 4.7 V resulted in a doubling of the heat dissipation when compared to charging to 4.3 V. A lowered chemical-to-electrical energy conversion efficiency due to thermal energy waste was observed, providing a complementary characterization of electrochemical degradation. The work demonstrates the utility of multi-modal X-ray and microcalorimetric approaches to understand the causes of capacity fade in lithium-ion batteries with Ni-rich NMC.

Ni-rich NMC is an attractive Li-ion battery cathode due to its combination of energy density, thermal stability, and reversibility. While higher delivered energy density can be achieved with a more positive charge voltage limit, this approach compromises sustained reversibility. Improved understanding of the local and bulk structural transformations as a function of charge voltage, and their associated impacts on capacity fade are critically needed. Through simultaneous operando synchrotron X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS) of cells cycled at 3-4.3 or 3-4.7 V, this study presents an in-depth investigation into the effects of voltage window on local coordination, bulk structure, and oxidation state. These measurements are complemented by ex situ X-ray fluorescence (XRF) mapping and scanning electrochemical microscopy mapping (SECM) of the negative electrode, X-ray photoelectron spectroscopy (XPS) of the positive electrode, and cell level electrochemical impedance spectroscopy (EIS). Initially, cycling between 3 and 4.7 V leads to greater delivered capacity due to greater lithium extraction, accompanied by increased structural distortion, moderately higher Ni oxidation, and substantially higher Co oxidation. Continued cycling at this high voltage results in suppressed Ni and Co redox, greater structural distortion, increased levels of transition metal dissolution, higher cell impedance, and 3× greater capacity fade.

The effects of a number of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines) upon the proliferation and the mitotic index of cultured L1210 cells and upon the survival of mice bearing P388 leukemia were determined. The 1,2-dihydrostructure and amino groups or masked amino groups at positions 5 and 7 were necessary for activity, and various substituents at positions 2 and 3 had considerable influence upon the activity. A number of these pyrazines had significant activity against i.p. P388 leukemia in mice, and several pyrazines were more active than the corresponding oxazines or thiazines in both the in vitro and the in vivo systems. The effects of the pyrazines upon the cultured cells were reversible, and the rate and degree of reversibility were influenced by the substituents at positions 2 and 3. Tests performed with two of the pyrazines yielded results that indicate that these compounds, like the known agent nocodazole, might compete with colchicine for binding to tubulin. Synergistic killing of cultured L1210 cells was obtained with combinations of one of the pyrazines and vincristine.

The effects of N,N'-bis(2-chloroethyl)-N-nitrosourea and chlorozotocin upon the proliferation, DNA synthesis, and viability of cultured cells of a sensitive line of L1210 leukemia, a line partially resistant to N,N'-bis(2-chloroethyl)-N-nitrosourea, and a line resistant to cyclophosphamide were determined. The results indicate that neither the effect upon proliferation nor the effect upon DNA synthesis is a good predictor of the extent of cell kill. The similarity of the effects of N,N'-bis(2-chloroethyl)-N-nitrosourea upon these two parameters for the three cell lines indicates that the sensitive and resistant cells are affected to approximately the same extent, but more of the resistant cells survive. Additional studies are required to seek the reasons for this differential survival.

Hydrolysis of the chloro group of ethyl (6-amino-4-chloro-5-nitropyridin-2-yl)carbamate (3) with formic acid gave the corresponding 4-hydroxypyridine 4. Catalytic hydrogenation of the nitro group of 4 gave the 5-amino-4-hydroxypyridine 5, which was reacted with alpha-halo ketones in acetic acid at room temperature to give a series of 3- and 2,3-substituted ethyl (5-amino-2H-pyrido[4,3-b][1,4]oxazin-7-yl)carbamates 8. Treatment of 8 with hot concentrated hydrochloric acid regenerated the pyridine synthon 5. In the reaction of 3 with thioacetate, the product underwent hydrolysis and air-oxidation to give the corresponding disulfide 6. Simultaneous reduction of both the nitro group and disulfide linkage of 6 gave the 5-amino-4-mercaptopyridine 7, which was reacted with alpha-halo ketones either in acetic acid at room temperature or in a mixture of ethanol and water at reflux to give a series of 3-, 2,3-, and 2,2,3-substituted ethyl (5-amino-2H-pyridol[4,3-b][1,4]thiazin-7-yl)carbamates 9. The effects of these pyridooxazines and pyridothiazines upon the proliferation and the mitotic index of cultured L1210 cells and upon the survival of mice bearing P388 leukemia were determined.

A rationale based on known chemical, biochemical, and biologic properties of nitrosoureas led to the testing of combinations of CCNU with chlorozotocin for in vivo activity against L1210 leukemia. Several combinations yielded synergistic antileukemic activity.

Ethyl 5-amino-1,2-dihydro-3-[N-methylanilino)methyl]pyrido[3,4-b]pyrazin-7-ylcarbamate (NSC 181928) is reported to be active against several experimental neoplasms. The experimental data obtained in the present study indicate that it causes the accumulation of cells at mitosis with both cultured cells and ascites cancer cells in vivo. This effect was observed with L1210, P388, colon cancer 26, colon cancer 38, and H.Ep. 2 cells in culture and with L1210 cells and P388 cells in mice. The agent was also active in vitro and in vivo against a line of leukemia P388 cells that are resistant to vincristine.

Certain derivatives containing the 1,2-dihydropyrido[3,4-b]pyrazine (1-deaza-7,8-dihydropteridine) ring system are active against experimental neoplasms in mice. The mechanism of action of these agents has been attributed to the accumulation of cells at mitosis. Identification of the structural features that are necessary for activity was accomplished by evaluation of modified 1-deazapteridines and ring and ring-opened analogues. Relative to ethyl 4-amino-1-deaza-7,8-dihydro-6-[(N-methylanilino)methyl]pteridine-2-carbamate (11) and the corresponding 6-phenyl compound (12), no antitumor activity was observed with 7,8-dihydropteridines, 3-deaza-7,8-dihydropteridines, and the corresponding heteroaromatic compounds. Also, activity was diminished or destroyed when 1-deaza-7,8-dihydropteridines were oxidized to 1-deazapteridines or reduced to 1-deaza-5,6,7,8-tetrahydropteridines. In addition, replacement of the 4-amino group with other substituents destroyed activity. The presence of a 6-substituent containing an aryl group appeared to be necessary for activity, which was increased when a methyl group was substituted at the 7-position.

Incubation at approximately physiological conditions of amino acids, peptides, and proteins with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or cyclohexyl isocyanate resulted in carbamoylation of the alpha-amino groups of amino acids, the terminal amino groups of peptides and proteins and the epsilon-amino groups of lysine moieties. Carbamoylation of the alpha-amino groups and the terminal amino groups occurred as readily as, or more readily than, the carbamoylation of the epsilon-amino groups. Carbamoylation of the amino groups of amino acids or peptides by 1,3-bis(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea altered the electrophoretic mobility of those compounds. Cyclization of (2-cloroethylcarbamoyl)-amino groups to form (2-oxazolin-2-yl)amino groups occurred at room temperature, and the resulting oxazolinyl compounds migrated electrophoretically similarly to the parent compounds. Since such cyclization did not occur with cyclohexylcarbamoylamino groups, treatment of amino acids, peptides, or proteins with 1,3-bis(2-chloroethyl)-1-nitrosourea might result in less permanent alteration of the respective charges on the resulting products than would treatment with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or other nitrosoureas lacking a 2-chloroethyl group on N-3. The relevance of these differences in charge to differences in physiological effects is not presently known. Although the present study does not establish a definite relationship between carbamoylation of any specific protein and the physiological effects of nitrosourea, it does reinforce and expand the existing evidence that carbamoylation of proteins is a proteins is a process that must be considered in efforts to explain the physiological effects of these agents, and it points to terminal amino groups of proteins as possible primary sites of carbamoylation.

Other investigators have reported that transplantable murine colon Tumor 26 is more sensitive than transplantable colon Tumor 38 to treatment with N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl)-N-nitrosourea. The present report presents the results of several kinds of in vivo and in vitro experiments that were performed to compare the effects of this agent upon these two tumors or upon cultured cells derived from them. In the in vivo experiments, data were also obtained for the spleens, colons, and marrow of the host animals. In the in vivo experiments, it was observed that: (a) approximately equal quantities of 14C from the 2-chloroethyl-14C-labeled agent were fixed to the DNA of the two tumors and the three host tissues following a single i.p. injection of the radioactive agent; (b) in all of the tissues examined at 24 hr after treatment, the drug caused greater inhibition of the synthesis of DNA than of the synthesis of RNA or protein, and the extents of inhibition of DNA synthesis were greater at 24 hr after treatment than at 6 hr after treatment; (c) the inhibition of DNA synthesis was slightly greater for Tumor 26 than for Tumor 38; (d) although the extents of inhibition of synthesis of DNA by Tumor 26 and by the colonic mucosa were similar at 24 hr after treatment of the animal, colonic mucosa much more quickly recovered the ability to synthesize DNA; and (e) the agent had no significant effect upon the sizes of the pools of purine and pyrimidine ribonucleoside phosphates. Cultured cells derived from the two tumors retained their tumorigenicity upon reimplantation into mice and their differential sensitivities to the agent, although approximately equal quantities of the 14C of the radioactive agent were fixed to the nuclei of the cells. In the in vitro experiments, the main effect of the agent upon the synthesis of macromolecules was the delayed inhibition of synthesis of DNA by Tumor 26 cells. Several experimental methods yielded evidence that the agent caused strand scission of the DNA of both kinds of cells, and there was more evidence of cross-linking of the DNA of Tumor 26 cells than of Tumor 38 cells. These results are consistent with the possibility that the differences in sensitivity of the two tumors to the agent are due to differences in the extents of cross-linking of the DNA. This explanation would be in agreement with the proposal suggested by other investigators who worked with other experimental systems.

Several properties of four 1-deaza-7,8-dihydropteridines were compared with those of each other and with those of colchicine, nocodazole, podophyllotoxin, and vincristine. Compound NSC 370147 was more active than the other compounds of this type with respect to inhibition of proliferation of cultured L1210 cells and to increase of the mitotic index. On an equimolar basis it was more active than two of the 1-deaza-7,8-dihydropteridines, colchicine, and nocodazole and was comparable to podophyllotoxin and vincristine in inhibiting the polymerization of partially purified pig brain tubulin. All four of the 1-deaza-7,8-dihydropteridines caused decreases in the extent of binding of [3H]colchicine to partially purified tubulin and enhanced the binding of [3H]vincristine to the tubulin. Emphasis in further testing was placed upon NSC 370147, because it is easier to synthesize and is more stable than some of the other compounds of this type and because its greater solubility in water facilitates its formulation for therapeutic administration. Compound NSC 370147 inhibited competitively the binding of [3H]colchicine to purified tubulin and enhanced slightly the binding of [3H]vincristine to tubulin. It was also synergistic with vincristine in killing cultured L1210 cells and in increasing the life-spans of mice bearing P388 leukemia. It is suggested that it would be worthwhile to evaluate combinations of NSC 370147 and vincristine in tests with other experimental neoplasms.

The effects of the 2-chloroethyl, 2-bromoethyl, and 2-fluoroethyl esters of (methylsulfonyl)methanesulfonic acid upon the DNA of cultured L1210 cells have been measured and compared with each other and with the effects of chlorozotocin. Results obtained by the alkaline elution method indicated that, at equimolar and equitoxic concentrations, the esters caused more strand scission than chlorozotocin, but at compound concentrations that caused a 50% reduction in colony formation by cells following an exposure period of 2 h, they caused no detectable cross-linking, whereas chlorozotocin did cause cross-linking. Two in vitro experimental methods that are based upon the complexing of ethidium to calf thymus DNA also yielded data showing that, at equimolar concentrations, chlorozotocin caused cross-linking of calf thymus DNA, but the 2-chloroethyl ester did not. These results indicate that these esters might not kill cells by producing DNA-DNA cross-links. The three esters caused qualitatively similar effects, but the fluoro esters caused less strand scission than the chloro and bromo esters, which caused about the same extent of strand scission.

Several nitrosoureidonucleosides (9a, 9b, 11a, 11b, 18 and 20) designed as inhibitors of enzymes that metabolize purine and pyrimidine nucleotides have been prepared and their chemical and biological properties studied. The low level of biological activity observed may be due to the unexpected stability of these nitrosoureas compared to biologically active compounds such as N-methyl-N-nitrosourea (MNU), N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), and N,N'-dicyclohexyl-N-nitrosourea (DCyNU).

The rates of incorporation of [C3H3]thymidine into the DNA of tissues of normal mice at various times after single doses of several drugs (adriamycin, vincristine, methotrexate, 5-fluorouracil [5-FU], melphalan, CCNU, and chlorozotocin) that have some clinical activity against cancer have been determined and are considered to be indicative of the "degree of normalcy" of the respective tissues at those times. These experiments show that there are some differences and some similarities in the effects of the agents upon the various tissues of the mouse. Each of the agents caused early inhibition of incorporation of [C3H3]thymidine followed by stimulation of such incorporation by intestinal mucosa and marrow, but the timing and the extents of inhibition and stimulation differed for the various agents. Except for CCNU, the preceding description would also apply to the effects upon liver and lungs, but the stimulation was delayed in comparison to that of intestinal mucosa and marrow. Early inhibition with little subsequent stimulation occurred in the spleen, kidneys, and heart. For all tissues, recovery was slower and less extensive after CCNU than after the other agents. The data obtained with the single agents were used for setting up experiments in which a single dose of one agent was followed after selected intervals of time with a single dose of a second agent. The combination of adriamycin plus 5-FU was less toxic when the two agents were given simultaneously than when the 5-FU was given 24-96 hours after the adriamycin, while for the combination of adriamycin plus CCNU there was less toxicity if the CCNU was administered 24 hours after the adriamycin rather than simultaneously with it. When a combination of a single dose of methotrexate and a single dose of 5-FU was administered to mice, the lethality was greater when the two agents were administered 120 hours apart than when the two agents were given simultaneously or were separated by 24 or 48 hours; this was true regardless of which agent was administered first. These results are consistent with what might be predicted upon the basis of the data obtained for the single agents in this study and of what is known about the mechanisms of action of these agents and of the effects of the agents in relation to cell and tissue kinetics. These results indicate that patterns of thymidine fixation by tissues following single doses of agents might be useful in scheduling multiple-drug therapy to minimize host toxicity.

Reaction of alpha-aminoacetophenone oximes (2) with ethyl 6-amino-4-chloro-5-nitropyridine-2-carbamate (1) gave ethyl 6-amino-5-nitro-4-[(2-oxo-2-phenylethyl)amino]pyridine-2-carbamate oximes (3), which were hydrolyzed under acidic conditions to give the corresponding ketones (4). Related pyridines substituted with a keto side chain were prepared from 1 and 1,3-diaminopropanone oximes and by oxidation of the side-chain hydroxy group of ethyl 6-amino-4- [[3-(N-methyl-N-phenylamino)-2-hydroxypropyl]amino]-5-nitropyridine-7- carbamates (6). Catalytic hydrogenation of the nitro group of 4 over Raney nickel in a large volume of ethanol gave the 1-deaza-7,8-dihydropteridines (7). Several of the oximes 3 were successfully hydrogenated to give 7 directly. The resulting 1-deaza-7,8-dihydropteridines showed potent cytotoxicity against cultured L1210 cells and significant anticancer activity against lymphocytic leukemia P-388 in mice. These biological activities are attributed to the accumulation of cells at mitosis.

The effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexl-1-nitrosourea, and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea on two nonmitochondrial DNA polymerases (I and II) purified from rat liver and hepatoma were examined. The activity of DNA polymerase I was not altered by treatment with any of the nitrosoureas or the corresponding isocyanates, 2-chloroethyl isocyanate and cyclohexyl isocyanate. Incubation of DNA polymerase II with the nitrosoureas (1 mM) inhibited its enzymatic activity 30 to 45%. DNA polymerase II was inhibited 75 and 90% by 1.mM 2-chloroethyl isocyanate and cyclohexyl isocyanate, respectively. The nitrosoureas appear to exert their inhabitory action on the enzyme (DNA polymerase II) rather than on the DNA template. Pretreatment of the enzyme increased the degree of inhibition by 1 mM nitrosourea (50 to 60% inhibition) or 2-chloroethul isocyanate (greater than 90% inhibition), whereas pretreatment of the DNA template did not enhance the inhibitory effect. The three nitrosoureas are equally effective as inhibitors of DNA polymerase II. 2-Chloroethyl isocyanate and cyclohexyl isocyanate are better inhibitors than are the nitrosoureas. Since further decomposition products of the isocyanates, 2-chloroethylamine and cyclohexylamine, do not inhibit DNA polymerase II, we conclude that the isocyanates, which are decomposition products of the nitrosoureas, are the active inhibitors of the enzyme.

Data for the alkylating activities, DNA cross-linking activities, and proliferation-inhibitory activities toward cultured L1210 cells for twenty-four 2-haloethyl sulfonates are reported. Previously reported activities against P388 leukemia in vivo are also presented to permit correlation of in vitro and in vivo properties. Since these compounds are believed to be 2-haloethylating agents, their properties and effects were compared with those of chlorozotocin, which is a recognized 2-chloroethylating agent. 2-Chloroethyl chloromethanesulfonate, which was the most effective compound against P388 leukemia, had a moderate level of alkylating activity and a low level of cross-linking activity, but it was quite active in inhibiting proliferation of cultured L1210 cells. Although its alkylating activity was about the same as that of chlorozotocin, it caused much less cross-linking of DNA. The in vitro tests were useful for gaining information relating structure to the individual properties, but results obtained for one of the properties might not be predictive of the relative values obtained for other properties nor for in vivo activity against P388 leukemia. These results indicate that additional experiments to define the mechanism of action of these agents are needed.

Alkylating anticancer drugs are varied in chemical structure, alkylating moieties, and likely mechanisms of cytotoxic activity for vital normal cells and sensitive tumor cells. This has been objectively documented by numerous examples illustrating: (1) different in vitro and in vivo reaction products; (2) greater than additive, additive, and less than additive cytotoxicity of drug combinations for vital normal cells in the mouse; (3) readily reproducible and often marked therapeutic synergism between a variety of 2-drug combinations of alkylating agents against a wide variety of histologic types of murine tumors, and (4) observed resistance and cross-resistance of a variety of murine tumors, selected for resistance to specific alkylating agents, compatible with recognized chemical and functional differences between these drugs. The most important observations on resistance and cross-resistance reported are: (a) L1210 cells selected for complete resistance to cyclophosphamide (CPA) retain full sensitivity to selected nitrosoureas (BCNU, CCNU, MeCCNU), chlorozotocin), dianhydrogalactitol, and cis-DDPt, while retaining marked but somewhat reduced sensitivity to L-PAM, piperazinedione, and thioTEPA. (B) L1210 cells selected for resistance to BCNU retain full sensitivity to CPA, L-PAM, and dianhydrogalactitol. They show complete cross-resistance to BIC and variable cross-resistance to other selected nitrosoureas and piperazinedione. (c) L1210/L-PAM has incomplete but marked resistance to L-PAM. It is similar to the parent drug-sensitive line (L1210/0) in response to BCNU, CCNU, MeCCNU, and BIC. It is variably (usually moderately) cross-resistant to CPA, chlorozotocin, dianhydrogalactitol, and thioTEPA, but is completely cross-resistant to cis-DDPt. These resistance and cross-resistance patterns, which are consistent with most other biological and chemical principles established with these alkylating agents, may be useful in selecting alkylating drug combinations for inclusion in chemotherapy protocols in man which, on the basis of diverse observations in animal tumor systems, appear to be clearly indicated.

Experimental results obtained with cultured L1210, human epidermoid No. 2, and Adenocarcinoma 755 cells are consistent in showing that inhibition of proliferation of the cells by 2,5-piperazinedione, 3,6-bis(5-chloro-2-piperidyl)-,dihydrochloride is accompanied by cell enlargement. Although cells initially in the G2 phase when exposure to the agent is begun can probably proceed through mitosis and divide, cells that are initially in G1 and S phases accumulate in the G2 phase. Progression of cells to G2 phase during the period of exposure to the agent is not a requisite for cell-killing, because cells exposed for periods insufficient to permit their accumulation in G2 are killed.