NEDD8-activating enzyme (NAE) (IC50 = 4.7 nM)

In purified enzyme assays that track the formation of E2-UBL thioester reaction products, pevonedistat (MLN4924) is a potent inhibitor of NAE and selective against the closely related enzymes UAE, SAE, UBA6, and ATG7 (IC50=1.5, 8.2, 1.8, and >10 μM, respectively). In pure enzyme and cellular assays, pevonedistat (MLN4924) preferentially inhibits NAE activity in contrast to the closely related ubiquitin-activating enzyme (UAE, also known as UBA1) and SUMO-activating enzyme (SAE; a heterodimer of SAE1 and UBA2 subunits). Strong cytotoxic activity is demonstrated by MLN4924 against several human tumor-derived cell lines[1].

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MLN4924 is a selective inhibitor of NAE [1]
MLN4924 was discovered as a result of iterative medicinal chemistry efforts on N6-benzyl adenosine that was originally identified as an inhibitor of NAE via high throughput screening (see Supplementary Information for chemical characterization). As shown in Fig. 1a, MLN4924 is structurally related to adenosine 5′-monophosphate (AMP)—a tight binding product of the NAE reaction. The main differences between AMP and MLN4924 are: (1) in place of the adenine base, MLN4924 has a deazapurine base substituted with an aminoindane at N6; (2) in place of the ribose sugar, MLN4924 has a carbocycle and the equivalent of the 2′-hydroxyl group of AMP is absent; (3) in place of the phosphate, MLN4924 has a sulphamate; and (4) in contrast to the stereochemistry of AMP, the methylene sulphamate of MLN4924 is in a non-natural anti-relationship to the deazapurine. X-ray crystallography confirmed that MLN4924 bound in the nucleotide-binding site of NAE.

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Through this approach, synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and TNF-α was identified. Pevonedistat is an inhibitor of the NEDD8-activating enzyme (NAE). Inhibition of NAE prevents activation of cullin-RING ligases, which are critical for proteasome-mediated protein degradation. TNF-α is a cytokine that is involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with the combination of pevonedistat and TNF-α, but not as single agents, resulted in rapid cell death. This cell death was determined to be mediated by caspase-8. Interestingly, the combination treatment of pevonedistat and TNF-α also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-α. Under conditions where apoptosis was blocked, the mechanism of death switched to necroptosis. [2]

In mice carrying HCT-116 xenografts, pevonedistat (MLN4924) (sc, 10 mg/kg, 30 mg/kg, or 60 mg/kg) inhibits the NEDD8 pathway, causing DNA damage[1]. The combination of pevonedistat (sc, 120 mg/kg) and TNF-α (10 μg/kg) damages the livers of SD rats[2].

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MLN4924 inhibits the NAE pathway in vivo [1]
To assess the ability of MLN4924 to inhibit NAE in vivo, HCT-116 tumour-bearing mice received a single subcutaneous dose of 10, 30 or 60 mg kg-1 MLN4924, and tumours were excised at various time-points over the subsequent 24 h period. The pharmacodynamic effects of treatment were assessed in tumour lysates which were analysed for NEDD8–cullin, NRF2 and CDT1 protein levels (Fig. 4a–c). A single dose of MLN4924 resulted in a dose- and time-dependent decrease of NEDD8–cullin levels as early as 30 min after administration of compound (Fig. 4a), with maximal effect 1–2 h post-dose. A significant difference was observed between the 10 and 60 mg kg-1 response profiles (P < 0.01), although the 10 and 30 mg kg-1 (P = 0.11) and 30 and 60 mg kg-1 (P = 0.24) profiles were not significantly different from each other. A single dose of MLN4924 also led to a dose- and time-dependent increase in the steady state levels of NRF2 and CDT1 (Fig. 4b, c). For all dose levels, NRF2 protein levels peaked 2–4 h after administration of MLN4924 and started to decline by 4–8 h post-dose. The timing of CDT1 accumulation was slightly delayed compared to NRF2, peaking 4 h after MLN4924 administration (Fig. 4c). Evidence of DNA damage in the tumour was indicated by the increased levels of phosphorylated CHK1 (Ser 317) at 8 h after a single administration of 30 and 60 mg kg-1 MLN4924 (Fig. 4d). It should be noted that MLN4924 also decreased NEDD8–cullin levels in normal mouse tissue as illustrated in mouse bone marrow cells (Supplementary Fig. 5). These data suggest that MLN4924-mediated inhibition of NAE in this in vivo tumour model results in pathway responses and cellular phenotypic effects compatible with those observed in cultured cells [1].

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Pevonedistat and TNF-α synergistically cause liver damage in rats [2]
The in vivo effects of pevonedistat and TNF-α were assessed in Sprague-Dawley rats. The dose of pevonedistat administered to rats was known from previous investigations to be well tolerated, and the dose of recombinant rat TNF-α activated TNF signaling without toxic side effects.4 Animals within each group (n=8) first received either vehicle or 10 μg/kg TNF-α, followed by either a second vehicle or 120 mg/kg pevonedistat 1 h later. Two animals dosed with the combination treatment exhibited moribund conditions and were euthanized within 10 h. There was a clear difference in liver damage of single-agent versus combination treatments in rats. The incidence and severity of microscopic liver findings for five representative animals from each dose group are presented in Table 1. The livers of animals dosed with pevonedistat+TNF-α had minimal-to-mild single-cell necrosis and neutrophilic infiltration. Representative histological images in Figure 6a illustrate karyomegaly (white arrowhead) in the livers from animals that received pevonedistat alone and necrosis (black arrowhead) and neutrophilic infiltrate (white arrow) in the combination-treated livers. Animals that received the combination treatment had significant ~5-fold elevation of the serum markers alanine transaminase (ALT), aspartate transaminase (AST) and sorbitol dehydrogenase (SDH) compared with those that received single-agent treatments (Figure 6b). Western blotting of liver extracts identified uncleaved caspase-8 (Figure 6c, arrow) in all animals and the p32 fragment of caspase-8 was observed in 9/10 animals that received pevonedistat±TNF-α (arrowhead). Neither p10 nor p18 (data not shown) were detected. Staining of the cleaved cFLIP-L 43-kDa fragment was strongest in samples that also had caspase-8 cleavage. There was a 4-fold elevation of caspase-8 activity in the pevonedistat±TNF-α groups compared with vehicle (Figure 6d). Whether caspase-8 activation was the principle driver of toxicity in rats could not be established.

In vitro E1-activating enzyme assays [1]
A time-resolved fluorescence energy transfer assay format was used to measure the in vitro activity of NAE. The enzymatic reaction, containing 50 μl 50 mM HEPES, pH 7.5, 0.05% BSA, 5 mM MgCl2, 20 μM ATP, 250 μM glutathione, 10 nM Ubc12–GST, 75 nM NEDD8–Flag and 0.3 nM recombinant human NAE enzyme, was incubated at 24 °C for 90 min in a 384-well plate, before termination with 25 μl of stop/detection buffer (0.1 M HEPES, pH 7.5, 0.05% Tween20, 20 mM EDTA, 410 mM KF, 0.53 nM Europium-Cryptate-labelled monoclonal Flag-M2-specific antibody and 8.125 μg ml-1 PHYCOLINK allophycocyanin (XL-APC)-labelled GST-specific antibody). After incubation for 2 h at 24 °C, the plate was read on the LJL Analyst HT Multi-Mode instrument using a time-resolved fluorescence method. A similar assay protocol was used to measure other E1 enzymes.

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Assay of bulk protein turnover [1]
HCT-116 cells were plated into 12-well plates at 1 × 105 cells per well and incubated overnight. The medium was exchanged with methionine-free DMEM containing 10% dialysed FBS and 50 μCi per well of [35S]methionine, and the cells were incubated for 20 min to label proteins undergoing synthesis. The cells were then washed three times with DMEM supplemented with 2 mM methionine. Fresh medium containing 10% FBS, 2 mM methionine and the test compounds as described in Fig. 2 were then added. At the specified time points, media (50 μl) was collected and subjected to liquid scintillation counting. At the end of the time course, remaining media was removed and the cells were solubilized by adding of 1 ml 0.2 N NaOH and the extract was subjected to liquid scintillation counting. The percentage of protein turnover at each time point was calculated as [(total acid soluble counts in supernatant)/(total acid soluble counts in supernatant + total counts in solubilized cells)] ×100.

Cell viability assay [1]
Cell suspensions were seeded at 3,000–8,000 cells per well in 96-well culture plates and incubated overnight at 37 °C. Compounds were added to the cells in complete growth media and incubated for 72 h at 37 °C. Cell number was quantified using the ATPlite assay.

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Western blot analysis of cultured cells [1]
HCT-116 cells grown in 6-well cell-culture dishes were treated with 0.1% DMSO (control) or MLN4924/pevonedistat for 24 h. Whole cell extracts were prepared and analysed by immunoblotting. For analysis of the E2–UBL thioester levels, lysates were fractionated by non-reducing SDS–PAGE and immunoblotted with polyclonal antibodies to Ubc12, Ubc9 and Ubc10. For analysis of other proteins, lysates were fractionated by reducing SDS–PAGE and probed with primary antibodies as follows: mouse monoclonal antibodies to CDT1, p27, geminin, ubiquitin, securin/PTTG and p53 or rabbit polyclonal antibodies to NRF2, Cyclin B1 and GADD34 . Rabbit monoclonal antibodies to NEDD8 and phosphorylated CHK1 (Ser 317) were generated by Millennium in collaboration with Epitomics, Inc. using Ac-KEIEIDIEPTDKVERIKERVEE-amide and Ac-VKYSS(pS)QPEPRT-amide as immunogens, respectively. Antibodies to pH3, cleaved PARP and cleaved caspase 3 were from Cell Signaling Technologies. Secondary HRP-labelled antibodies to rabbit IgG or mouse IgG (Santa Cruz) were used as appropriate. Blots were developed with ECL reagent. For Supplementary Fig. 2, the secondary antibody was Alexa-680-labelled antibody to rabbit/mouse IgG and the blots were imaged using the Li-Cor Odyssey Infrared Imaging system.

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Cell-cycle analysis [1]
Logarithmically growing HCT-116 cells were incubated with either MLN4924/pevonedistat or DMSO for the times indicated. Collected cells were fixed in 70% ethanol and stored overnight at 4 °C. Fixed cells were centrifuged to remove ethanol, and the pellets were resuspended in propidium iodide and RNase A in PBS for 1 h on ice protected from light. Cell-cycle distributions were determined using flow cytometry and analysed using Winlist software (Verity).

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FACS analysis [2]
DNA nuclear content was determined as previously described.15 Actively dividing H-4-II-E cells were treated with MLN4924/pevonedistat and/or TNF-α for 8 h. Before the end of treatment, cells were spiked with 10 μM bromodeoxyuridine (Brd-U). After 30 min, cells were fixed in ethanol, incubated with a FITC–anti-Brd-U secondary antibody, and then incubated with 10 μg/ml propidium iodide (PI). Labeled cells were measured for Brd-U and PI staining on a FACSCalibur flow cytometer. Cell cycle data were analyzed using FACSDiva (v 6.1.1).

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siRNA knockdown [2]
H-4-II-E cells were transfected with either a non-targeting control pool of siRNAs or with individual siGenome siRNA oligonucleotide duplexes designed to silence target rat genes caspase-8 and cdt1. Cells were plated sparsely (10 000 cells/well in 96-well plates and 500 000 cells/well in a six-well tissue-culture plate) in antibiotic-free media. The following day, cells were transfected with 25 nM of siRNAs using Lipofectamine RNAiMAX for 72 h. Following transfection, cells were treated with MLN4924/pevonedistat and/or TNF-α for 24–48 h. Successful knockdown were verified by western blotting. Sequences for siRNAs used in experiments are included in Supplementary Information.

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Cell culture [2]
The rat hepatoma H-4-II-E cell line was selected to model MLN4924/pevonedistat toxicities because of its common use in the assessment of toxic compounds.43,44 H-4-II-E cells were purchased from American Type Culture Collection and were cultured following the manufacturer’s instructions. Briefly, cells were cultured in MEM supplemented with 10% FBS and incubated at 37 °C with 5% CO2. For routine culture, cells were supplemented with 10 U/l of penicillin and 10 ug/l of streptomycin. For passaging, cells were washed once with PBS, treated with 0.05% trypsin-EDTA, supplemented with fresh media, and pelleted in a clinical centrifuge.

Tumour xenograft efficacy experiments [1]
Female athymic NCR mice were used in all in vivo studies. All animals were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals. Mice were inoculated with 2 × 106 HCT-116 cells (or 30–40 mg H522 tumour fragments) subcutaneously in the right flank, and tumour growth was monitored with caliper measurements. When the mean tumour volume reached approximately 200 mm3, animals were dosed subcutaneously with vehicle (10% cyclodextrin) or MLN4924. Inhibition of tumour growth (T/C) was calculated on the last day of treatment.

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Pharmacodynamic marker analysis [1]
Mice bearing HCT-116 tumours of 300–500 mm3 were administered a single MLN4924 dose, and at the indicated times tumours were excised and extracts prepared. The relative levels of NEDD8–cullin and NRF2 were estimated by quantitative immunoblot analysis using Alexa680-labelled anti-IgG (Molecular Probes) as the secondary antibody. The statistical difference between the groups for NEDD8–cullin inhibition was determined using the Kruskal–Wallis test. For the analysis of CDT1 and phosphorylated CHK1 (Ser 317) levels in tumour sections, formalin-fixed, paraffin-embedded tumour sections were stained with the relevant antibodies, amplified with HRP-labelled secondary antibodies and detected with the ChromoMap DAB Kit). Slides were counterstained with haematoxylin. Images were captured using an Eclipse E800 microscope and Retiga EXi colour digital camera and processed using Metamorph software. CDT1 and phosphorylated CHK1 levels are expressed as a function of the DAB signal area.

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Isolation of bone marrow cells from mice [1]
For bone marrow pharmacodynamic studies, naive NCr-Nude mice were administered MLN4924, and at the indicated times leg bones were excised. Marrow was flushed from the bones with PBS, pelleted by centrifugation and flash frozen. Thawed marrow was lysed in M-PER buffer (Pierce) with protease inhibitors. NEDD8–cullin levels were measured by immunoblot analysis.

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In vivo rat model [2]
Ten-week-old male Sprague-Dawley rats were used. Across two studies, a total of eight animals in each group were dosed with vehicle, TNF-α, pevonedistat, or pevonedistat+TNF-α. Animals were first intravenously administered either vehicle (1× PBS) or 10 μg/kg TNF-α. One hour later, they were subcutaneously administered vehicle (20% sulfobutyl ether beta-cyclodextrin in 50 mM citrate buffer, pH 3.3) or 120 mg/kg pevonedistat. Scheduled euthanasia occurred 24 h postdose. Unscheduled euthanasia was performed when animals exhibited moribund conditions. Serum was collected at necropsy and analyzed for serum chemistry markers of liver damage (ALT, AST, and SDH). Additionally, the livers from five animals in each group were removed, separated into two sections and either frozen at −80 °C for subsequent protein analysis or fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4–6 μm, mounted on glass slides, stained with hematoxylin and eosin, and analyzed with an Olympus BX51 light microscop for histopathology assessment. Microscopic findings were recorded in concordance with the standardized nomenclature for classifying lesions within the livers of rats.

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10% cyclodextrin;60 mg/kg;Subcutaneously injection

mice bearing HCT-116 xenografts

[1]. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009 Apr 9;458(7239):732-6.

[2]. The NAE inhibitor pevonedistat (MLN4924) synergizes with TNF-α to activate apoptosis. Cell Death Discovery 1, Article number: 15034 (2015).

Pevonedistat is a pyrrolopyrimidine that is 7H-pyrrolo[2,3-d]pyrimidine which is substituted by a (1S)-2,3-dihydro-1H-inden-1-ylnitrilo group at position 4 and by a (1S,3S,4S)-3-hydroxy-4-[(sulfamoyloxy)methyl]cyclopentyl group at position 7. It is a potent and selective NEDD8-activating enzyme inhibitor with an IC50 of 4.7 nM, and currently under clinical investigation for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes. It has a role as an apoptosis inducer and an antineoplastic agent. It is a pyrrolopyrimidine, a secondary amino compound, a member of cyclopentanols, a sulfamidate and a member of indanes.
Pevonedistat has been used in trials studying the treatment of Lymphoma, Solid Tumors, Multiple Myeloma, Hodgkin Lymphoma, and Metastatic Melanoma, among others.
Pevonedistat is a small molecule inhibitor of Nedd8 activating enzyme (NAE) with potential antineoplastic activity. Pevonedistat binds to and inhibits NAE, which may result in the inhibition of tumor cell proliferation and survival. NAE activates Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8), an ubiquitin-like (UBL) protein that modifies cellular targets in a pathway that is parallel to but distinct from the ubiquitin-proteasome pathway (UPP). Functioning in diverse regulatory activities, proteins conjugated to UBLs like Nedd8 typically are not targeted for proteasomal degradation.

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Drug Indication
Treatment of acute myeloid leukaemia, Treatment of myelodysplastic syndromes.

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The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.[1]

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Here we have described the initial characterization of MLN4924, a small molecule inhibitor of NAE that represents a new approach to targeting the UPS for the treatment of cancer. MLN4924 completely inhibits detectable NAE pathway function in cells, disrupting the turnover of CRL substrates, with important roles in cell-cycle progression and survival. Our results indicate that inhibition of the NAE pathway disrupts cancer cell protein homeostasis more selectively than the inhibition of proteasome activity, which may contribute to useful differences in clinical efficacy and safety profiles. Sustained NAE pathway inhibition was found to result in the activation of apoptosis as a consequence of cell-cycle-dependent DNA re-replication. This phenotype was presumably a result of the inability of the cell to degrade the CRL substrate CDT1, which has been shown to induce re-replication when overexpressed. Similar cell-cycle profiles were obtained when NAE levels were reduced by RNAi or when NAE activity was compromised in a temperature-sensitive mutant cell line.
In vivo, we demonstrated that MLN4924 suppressed the growth of human tumour xenografts at doses and schedules that were well tolerated. Analysis of tumours from treated animals confirmed inhibition of the NEDD8 pathway, suggesting that these pharmacodynamic markers may have use in monitoring NAE inhibition in patients treated with MLN4924. These preclinical findings have supported the transition of MLN4924 into clinical development. [1]

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Predicting and understanding the mechanism of drug-induced toxicity is one of the primary goals of drug development. It has been hypothesized that inflammation may have a synergistic role in this process. Cell-based models provide an easily manipulated system to investigate this type of drug toxicity. Several groups have attempted to reproduce in vivo toxicity with combination treatment of pharmacological agents and inflammatory cytokines. Through this approach, synergistic cytotoxicity between the investigational agent pevonedistat (MLN4924) and TNF-α was identified. Pevonedistat is an inhibitor of the NEDD8-activating enzyme (NAE). Inhibition of NAE prevents activation of cullin-RING ligases, which are critical for proteasome-mediated protein degradation. TNF-α is a cytokine that is involved in inflammatory responses and cell death, among other biological functions. Treatment of cultured cells with the combination of pevonedistat and TNF-α, but not as single agents, resulted in rapid cell death. This cell death was determined to be mediated by caspase-8. Interestingly, the combination treatment of pevonedistat and TNF-α also caused an accumulation of the p10 protease subunit of caspase-8 that was not observed with cytotoxic doses of TNF-α. Under conditions where apoptosis was blocked, the mechanism of death switched to necroptosis. Trimerized MLKL was verified as a biomarker of necroptotic cell death. The synergistic toxicity of pevonedistat and elevated TNF-α was also demonstrated by in vivo rat studies. Only the combination treatment resulted in elevated serum markers of liver damage and single-cell hepatocyte necrosis. Taken together, the results of this work have characterized a novel synergistic toxicity driven by pevonedistat and TNF-α.[2]

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The results of this study demonstrate that the combination of the NAE inhibitor pevonedistat and the pro-inflammatory cytokine TNF-α is toxic. The driver of in vitro toxicity appears to be enhanced cleavage/activation of the caspase-8 p10 protease, which in turn activated apoptosis. However, the molecular mechanism that links pevonedistat to caspase-8 remains unclear in the pevonedistat and TNF-α cytotoxicity model. As cullin-3 can ubiquitinate caspase-8 (Jin et al.36) and is also inhibited by pevonedistat, it was an obvious candidate for investigation, but cullin-3 knockdown did not increase sensitivity to single-agent TNF-α (Supplementary Figure S4). Ultimately, a role for cullin-3 in mediating the synergistic toxicity was not established. Single-agent pevonedistat is known to stabilize the expression of ≥120 different proteins,42 none of which are known to interact with caspase-8. A higher-throughput approach is needed to determine if any unrecognized proteins become stabilized in response to pevonedistat+TNF stimulation. Further investigations using pevonedistat as a tool compound will lead to a better understanding of the molecular mechanisms that underlie programmed cell death.[2]

C21H25N5O4S

443.52

443.162

C, 56.87; H, 5.68; N, 15.79; O, 14.43; S, 7.23

905579-51-3

Pevonedistat hydrochloride;1160295-21-5

16720766

White to light yellow solid powder

1.6±0.1 g/cm3

721.0±70.0 °C at 760 mmHg

161-163°C

389.9±35.7 °C

0.0±2.4 mmHg at 25°C

1.769

2.16

3

8

6

31

734

4

C1CC2=CC=CC=C2[C@H]1NC3=C4C=CN(C4=NC=N3)[C@@H]5C[C@H]([C@H](C5)O)COS(=O)(=O)N

MPUQHZXIXSTTDU-QXGSTGNESA-N

InChI=1S/C21H25N5O4S/c22-31(28,29)30-11-14-9-15(10-19(14)27)26-8-7-17-20(23-12-24-21(17)26)25-18-6-5-13-3-1-2-4-16(13)18/h1-4,7-8,12,14-15,18-19,27H,5-6,9-11H2,(H2,22,28,29)(H,23,24,25)/t14-,15+,18-,19-/m0/s1

[(1S,2S,4R)-4-[4-[[(1S)-2,3-dihydro-1H-inden-1-yl]amino]pyrrolo[2,3-d]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl sulfamate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 5 mg/mL (11.27 mM) (saturation unknown) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.64 mM) (saturation unknown) in 5% DMSO + 95% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.69 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (4.69 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (4.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 1% DMSO 99% Saline

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 (Please use freshly prepared in vivo formulations for optimal results.)