This publication is Open Access under the license indicated. Learn More
Drug Annotation
Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging VirusesClick to copy article linkArticle link copied!
- Dustin SiegelDustin SiegelGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Dustin Siegel
- Hon C. Hui
- Edward DoerfflerEdward DoerfflerGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Edward Doerffler
- Michael O. ClarkeMichael O. ClarkeGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Michael O. Clarke
- Kwon Chun
- Lijun Zhang
- Sean Neville
- Ernest Carra
- Willard Lew
- Bruce Ross
- Queenie Wang
- Lydia Wolfe
- Robert JordanRobert JordanGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Robert Jordan
- Veronica SolovevaVeronica SolovevaUnited States Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick, Maryland 21702, United StatesMore by Veronica Soloveva
- John Knox
- Jason Perry
- Michel PerronMichel PerronGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Michel Perron
- Kirsten M. StrayKirsten M. StrayGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Kirsten M. Stray
- Ona BarauskasOna BarauskasGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Ona Barauskas
- Joy Y. Feng
- Yili Xu
- Gary Lee
- Arnold L. RheingoldArnold L. RheingoldUniversity of California—San Diego, San Diego, California 92093, United StatesMore by Arnold L. Rheingold
- Adrian S. RayAdrian S. RayGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Adrian S. Ray
- Roy BannisterRoy BannisterGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Roy Bannister
- Robert StrickleyRobert StrickleyGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Robert Strickley
- Swami SwaminathanSwami SwaminathanGilead Sciences, Inc., Foster City, California 94404, United StatesMore by Swami Swaminathan
- William A. LeeWilliam A. LeeGilead Sciences, Inc., Foster City, California 94404, United StatesMore by William A. Lee
- Sina BavariSina BavariUnited States Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick, Maryland 21702, United StatesMore by Sina Bavari
- Tomas Cihlar
- Michael K. LoMichael K. LoCenters for Disease Control and Prevention, Atlanta, Georgia 30333, United StatesMore by Michael K. Lo
- Travis K. WarrenTravis K. WarrenUnited States Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick, Maryland 21702, United StatesMore by Travis K. Warren
- Richard L. Mackman*Richard L. Mackman*E-mail: [email protected]. Tel.: 650 522 5258.Gilead Sciences, Inc., Foster City, California 94404, United StatesMore by Richard L. Mackman
Abstract
Click to copy section linkSection link copied!
The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1′-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3–14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016, 531, 381−385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.
Copyright © 2017 American Chemical Society
Note
This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
Introduction
Click to copy section linkSection link copied!
Ebola virus disease (EVD) was first documented 40 years ago during an outbreak of infectious hemorrhagic fever in Northern Zaire (current Democratic Republic of Congo). More than 20 intermittent outbreaks have occurred since then, but the most recent outbreak in West Africa spanning 2013–2016 has been the largest recorded in history and presented an international public health emergency. (1) Over 28,000 cases were confirmed in Guinea, Liberia, and Sierra Leone resulting in >11,000 deaths including >500 healthcare workers, which severely strained the local medical infrastructure. (2) In survivors, the Ebola virus (EBOV) can persist in bodily fluids for months after the onset of acute infection potentially leading to EVD-related sequelae and viral recrudescence. (3) While rare, secondary transmission has been documented to occur through sexual intercourse implicating persistent virus in genital secretions. (4) Despite the end of the current outbreak, the potential for equally devastating future outbreaks together with the persistent virus observed in survivors makes the development of a safe, effective, and readily available treatment option for EVD a high priority.
EBOV, a member of the Filoviridae family, is a single-stranded, negative-sense, nonsegmented RNA virus that is the causative agent of EVD. Other Filoviridae family members include Marburg, Sudan, and Bundibugyo viruses, which have all been responsible for outbreaks associated with high mortality rates in sub-Saharan Africa. (5,6) Over the course of the recent West African EVD outbreak, several direct acting anti-Ebola agents including monoclonal antibodies (ZMapp), (7) interfering-RNAs, (8−10) and small molecule nucleoside(tide) antivirals such as favipiravir (1), (11−13) and brincidofovir (2) (14) have been evaluated in early clinical trials (Figure 1). More recently another nucleoside analogue, galidesivir (3, BCX4430 (15)), has entered clinical development. These developments are encouraging, but to date, none of these potential therapeutics have established robust clinical efficacy for the treatment of acute infection or the viral persistence and sequelae. Several vaccines have shown strong promise for preventing EBOV infection, but the breadth and durability of protection they can afford has yet to be established. (16)
Figure 1
Figure 1. Structures of antiviral nucleosides and nucleoside phosphonates.
Prior to the Ebola outbreak, we had embarked on a strategic initiative aimed at evaluating the potential of nucleoside analogues for the treatment of selected emerging viruses. A library of ∼1000 diverse nucleoside and nucleoside phosphonate analogues was harnessed from over 2 decades of research across multiple antiviral programs. In collaboration with the Center for Disease Control and Prevention (CDC) and the United States Army Medical Research Institute of Infectious Diseases (USAMRIID), selected compounds from the library were screened against EBOV, leading to the identification of parent 4 and a potent monophosphate prodrug mixture 4a that contained the single Sp isomer 4b (GS-5734 (17)) that was selected for development. This report describes in detail the structure activity relationships (SAR) of the parent nucleoside, prodrug optimization and selection, and synthesis optimization of the development candidate 4b. Candidate compound 4b is currently in phase 2 trials to assess the effect on the chronic shedding of virus in EVD survivors following promising efficacy data established in a non-human-primate (NHP) EVD challenge model. These data have been recently reported (17) and will be summarized along with the early clinical experience with 4b.
Results and Discussion
Click to copy section linkSection link copied!
The assembly of the ∼1000 compound nucleos(t)ide screening library was heavily focused toward ribose analogues that could target RNA viruses since this would encompass many emerging viral infections ranging from respiratory pathogens belonging to the Coronaviridae family such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), to mosquito-borne viruses of the Flaviviridae family such as Dengue and Zika. The majority of the library compounds were nucleosides that contained a cyclic modified ribose or “ribose-like” core. These nucleosides were also predominantly N-nucleosides. Less than 10% of the library comprised nucleoside phosphonates or acyclic analogues due to the limited success, to date, in identifying potent RNA virus inhibitors with these types of analogs. A second key factor in the library assembly was that approximately 50% of the library included monophosphate and ester prodrugs to capture analogs that may be missed in cellular screens due to either poor permeability or inefficient metabolism in the respective cell types that the different antiviral assays utilize. Nucleoside analogs require activation by intracellular nucleoside/tide kinases to generate their respective nucleoside triphosphate (NTP) metabolites in order to then compete with endogenous natural nucleotide pools for incorporation into the replicating viral RNA. (17) The first phosphorylation step to generate the nucleoside monophosphate is often rate limiting, and therefore the application of monophosphate prodrugs, especially phosphoramidates (ProTides), has been extensively explored in nucleoside analogs to bypass this initial phosphorylation step. (18) A notable example includes the phosphoramidate prodrug Sofosbuvir (5) for the treatment of HCV (Figure 1). (19) Nucleoside phosphonate analogs are bioisosteres of the monophosphates but also require prodrugs to enable masking of the charged phosphonate acid thereby allowing more efficient entry into cells. A recent example of an approved drug in this class is the phosphonamidate prodrug tenofovir alafenamide (6) for the treatment of HIV. (20) In both examples the amidate prodrugs effectively deliver high levels of triphosphate (diphosphophosphonate in the case of nucleoside phosphonates) inside the target cells and demonstrate significant improvements in potency compared to their respective parent nucleos(t)ides when screened in antiviral assays. (20,21)
In the original library screening toward a panel of RNA viruses across different viral families, promising leads were identified. Subsequent to the EVD outbreak, some of these analogs were selected for EBOV testing in collaboration with the CDC and USAMRIID in a BSL-4 facility. From this screen nucleoside 4, (22) a 1′-CN modified adenosine C-nucleoside emerged with sub-micromolar activity toward EBOV in human microvascular endothelial cells (HMVEC-TERT) cells (entry 2, Table 1). In addition, its phosphoramidate prodrug mixture 4a (23) (entry 3, Table 1) containing ∼1:1 ratio of Sp 4b and Rp 4c diastereoisomers (Figure 1) was found to be very potent toward EBOV in both HeLa and HMVEC cells. Encouraged by these data, the anti-EBOV activity for a range of nucleoside analogs and their prodrugs was evaluated and the results are reported in Table 1, along with activity toward respiratory syncytial virus (RSV), from the Pneumoviridae family, and HCV, from the Flaviviridae family.
Table 1. SAR of Nucleoside Parents and Selected Prodrugsa
| entry | compd | EBOV EC50 HeLa cells (μM) | EBOV EC50 HMVEC cellsb (μM) | RSV EC50 HEp-2 cells (μM) | HCV 1b EC50 Huh-7 cells (μM) | CC50 HEp-2 cells (μM) | CC50 Huh-7 cells (μM) | CC50 MT4 cells (μM) |
|---|---|---|---|---|---|---|---|---|
| 1 | 7 | 0.0035 | 0.038c | 0.15 | <0.01 | |||
| 2 | 4 | >20 | 0.78 | 0.53 | 4.1 | >100 | >88 | >57 |
| 3 | 4a | 0.17 | 0.12 | 0.027 | 0.023d | 9.2 | 17d | 2.0 |
| 4 | 4b | 0.10 | 0.053 | 0.015 | 0.057 | 6.1 | 36 | 1.7 |
| 5 | 8 | >10 | 5.5 | 38 | 93 | 62 | 4.5 | |
| 6 | 9 | >200 | >88 | >200 | >88 | 120 | ||
| 7 | 9a | 3.9 | 1.1 | 6.9 | >100 | >44 | >32 | |
| 8 | 10 | 56 | >100 | >44 | >100 | >88 | >53 | |
| 9 | 11 | >50 | >10 | 7.3 | 12 | >100 | >44 | >57 |
| 10 | 11a | >20 | 63 | 2.5 | >100 | >44 | 53 | |
| 11 | 12 | >100c | >44 | >44 | 32c | |||
| 12 | 13 | 50 | >10 | >100 | >44 | >100 | >44 | >57 |
| 13 | 13a | 27 | 13d | >50 | 0.37 | >50 | >44 | 1.4 |
| 14 | 13b | >20 | 40 | >20 | 0.31 | 95 | 51 | 7.8 |
a
Data reported are at least n ≥ 2 in 384 well assay format unless otherwise noted.
b
HMVEC cells = TERT-immortalized human foreskin microvascular endothelial cells (ATCC-4025) cells.
c
96 well assay format.
d
n = 1 data only.
The presence of the 1′-CN modification in 4 was found to be critical in providing selectivity toward viral polymerases and avoiding the significant toxicity (CC50 < 0.01–0.15 μM) associated with the unmodified C-nucleoside 7 (entry 1, Table 1). (24) The MT4 cell line was also used as a sensitive cell line to evaluate cytostatic effects of nucleoside analogs and confirmed the poor selectivity of 7 observed in both the HEp-2 and Huh-7 cell lines. The prodrug mixture 4a, in addition to potent anti-EBOV activity, demonstrated significant activity toward RSV and HCV, with potencies similar to or better than that for EBOV (EC50 < 120 nM). The broad and potent antiviral activity across all three viruses for 4a was further supported by the potent activity of the single Sp isomer 4b toward the same viruses and also other emerging RNA viruses such as MERS and Junin viruses, and to a lesser extent Lassa. (17) The antiviral selectivity of 4b toward EBOV was 17–32-fold compared to the MT4 cell line CC50, and higher in the other cell types reported in Table 1. Given the anticipated short treatment duration for EVD, this window of in vitro selectivity was considered sufficient for continued interest in 4b. The 1′-methyl analogue 8 (entry 5) (22) was less active toward EBOV and also displayed a higher degree of toxicity compared to the 1′-CN analogue 4 illustrating how small changes in the polarity and size of the 1′ substituent can impact the overall profile. The 1′-ethynyl analogue 9 (entry 6) (22) and its corresponding 2-ethylbutyl alanine prodrug 9a (entry 7) were both less active when compared to their respective 1′-CN counterparts (4 and 4a, respectively).
Compound 4 is a C-nucleoside analogue which provides chemical and enzymatic stability toward deglycosylation reactions at the anomeric center. However, alternate base modifications including N-nucleosides were also studied. Interestingly, the corresponding 1′-CN modified adenosine N-analogue 10 (entry 8) (25) was significantly less active toward all viruses, while the 1′-CN modified N-nucleoside pyrimidine 11 (entry 9) retained weak antiviral activity only for RSV and HCV. (26,27) The phosphoramidate prodrug of 1′-CN cytidine 11a (entry 10) (27) did not improve the potency toward EBOV (in HeLa cell assay) or the other viruses tested, presumably due to limitations in metabolism beyond the monophosphate. In general, the potency trends of 1′ substitution and nucleobase changes were similar across EBOV, RSV, and HCV, which
To interrogate the cell based SAR more rigorously the active NTP metabolite 4tp (23) was tested toward the viral polymerases (Table 2). The triphosphate 4tp demonstrated a half-maximal inhibitory concentration (IC50) of 1.1 μM against the RSV RdRp and 5.0 μM against HCV RdRp. The Ebola viral polymerase has to date evaded efforts toward its isolation and expression, so the intrinsic activity of the active NTP metabolite cannot be directly evaluated. An alternate method for estimating the inhibitory properties of an NTP for its viral target is to measure the NTP levels inside cells following incubation with the parent or prodrug compound at a given concentration and then calculate the NTP levels at the EC50 measured in the same cells. (17) For example, in a continuous 72 h incubation of 1 μM 4a, the 4tp levels were measured at 2, 24, 48, and 72 h, and reached a Cmax of 300, 110, and 90 pmol/million cells in macrophages, HMVEC, and HeLa cells lines, respectively. The several-fold difference in maximum 4tp levels is not unusual and reflects the differences between cells with respect to their ability to break down the prodrug and subsequently metabolize the released monophosphate to the active 4tp. The average NTP levels over the 72 h incubation of 4a were then used along with an average cell volume of 2 pL (29) to calculate an estimated half-maximal inhibitory concentration of ∼5 μM for the intracellular inhibition of EBOV polymerase. This is comparable in potency toward RSV and HCV polymerases supporting the potent antiviral EC50 data demonstrated for the prodrug mixture 4a across the three viruses when allowing for cell differences (Table 1). The selective inhibition of the viral polymerases vs host polymerases is considered a key factor in the development of a safe and effective nucleoside antiviral. (30,31) Therefore, 4tp was evaluated toward several host polymerases and was found to be a weak incorporator toward mitochondrial polymerase (POLRMT) and not a substrate for DNA polymerase γ, as would be expected given the presence of the ribose 2′ OH (Table 2). Across the host RNA and DNA polymerases evaluated there was no inhibition up to 200 μM (Table 2) demonstrating a high degree of selectivity of 4tp toward the viral polymerases compared to representative examples of host polymerases.
Table 2. Inhibition of RSV Polymerase, HCV Polymerase, and Human Polymerases by 4tp
| enzyme | 4tp IC50 (μM) | 4tp SNIa rate (%) |
|---|---|---|
| RSV RdRp | 1.1 | |
| HCV RdRp | 5 | |
| POLRMT | >200 | 6 |
| RNA Pol II | >200 | |
| DNA Pol α | >200 | |
| DNA Pol β | >200 | |
| DNA Pol γ | >200 | 0 |
a
SNI = single nucleotide incorporation.
Molecular structure information is not available for the EBOV or RSV polymerases, so modeling of the active sites was performed based on the published structures for HIV and HCV polymerases, together with an analysis of the respective sequences. (32) Within the modeled active site of EBOV polymerase the major difference between EBOV and RSV is Y636 (EBOV) compared to F704 (RSV) and the major difference between EBOV and HCV is E709 (EBOV) compared to S282 (HCV). On docking the triphosphate 4tp, the 1′-CN group occupies a pocket formed by residues that are identical between EBOV and RSV, yet very different in HCV (Figure 2a). Nevertheless the 1′-CN analogue retains antiviral potency across these viruses and others (17) suggesting that a pocket exists to accommodate the 1′-CN group in many viral polymerases including other filoviruses. (33) The 2′-β-H of 4tp is in close proximity to E709, and replacement of this group with a 2′-β-methyl (13tp) would be anticipated to interfere with E709 (Figure 2b). (34) However, the 2′-β-methyl can be accommodated by the larger pocket afforded by the smaller S282 residue of HCV (Figure 2c). This suggests the lack of activity for 2′-β-methyl analogs toward EBOV and RSV and retained potency toward HCV is likely due to steric constraints in the polymerase active site. Consistent with the model, EBOV and RSV both have the E709 or equivalent residue, and 13tp was found to be significantly less active (IC50 > 30 μM) toward RSV.
Figure 2
Figure 2. (a) Compound 4tp modeled into the EBOV polymerase active site. Residue Y636 is highlighted in green surface, sits below the ribose, and corresponds to F704 in RSV. Residue E709 is highlighted in red surface, sits in proximity to the 2′-β-H position of the ribose, and corresponds to S282 in HCV. (b) Compound 13tp modeled into the EBOV polymerase active site. The 2′-β-methyl overlaps with residue E709 highlighted in red. (c) Compound 13tp modeled into the HCV polymerase active site. Residue S282 is highlighted in the yellow surface, and the 2′-β-methyl can be accommodated.
The screening and modeling efforts established 4 as the best lead for prodrug optimization. The ability to evaluate prodrugs, especially in vivo, required an efficient synthesis route for both the nucleoside 4 and, preferably, a single prodrug diastereoisomer. Neither was available at the outset, so significant chemistry resources were applied to improve the robustness and scalability of the route along with generation of single prodrug diastereoisomers. The first generation synthesis of 4 and the single Sp phosphoramidate prodrug 4b commenced with a glycosylation reaction via metal–halogen exchange of the bromo-base 15 followed by addition into the ribolactone 14 (Scheme 1). Two conditions were identified to render this desired C–C bond formation. The first condition (a) proceeded through addition of excess n-BuLi to a mixture of TMSCl and 15, which was designed to result in lithium–halogen exchange after removal of the acidic 6N protons by silyl protection. Addition of this in situ generated reagent to the ribolactone 14 then afforded 16 in 25% yield. (23,35,36) The alternative conditions (b) employed sodium hydride and 1,2-bis(chlorodimethylsilyl)ethane for the 6N protection step, followed by lithium–halogen exchange, and addition to the lactone to afford 16 in 60% yield. (22,36)
Scheme 1
Scheme 1. First Generation Synthesis of 4ba
aReagents and conditions: (a) n-BuLi, (TMS)Cl, THF, – 78 °C, 25%; (b) 1,2-bis(chlorodimethylsilyl)ethane, NaH, n-BuLi, THF, – 78 °C, 60%; (c) (TMS)CN, BF3·Et2O, CH2Cl2, – 78 °C, 58% (89:11β-17/α); (d) BCl3, CH2Cl2, – 78 °C, 74%; (e) 19, NMI, OP(OMe)3, 21%; (f) OP(OPh)Cl2, Et3N, CH2Cl2, 0 °C, 23%.
The efficiency of both conditions was suboptimal as the yields were capricious and highly dependent on the cryogenic temperatures and the rate of n-BuLi addition required for the transformation. Furthermore, premature quenching and reduction of lithio base was observed, which was rationalized to be a consequence of deprotonation α to the lactone under the highly basic conditions. Compound 16 was isolated as a mixture of 1′-isomers, which were taken into the subsequent 1′-cyanation reaction to isolate the major product, β-anomer 17, by chromatography. (37) Following removal of the three benzyl protecting groups to afford 4, the diastereomeric mixture of the phosphoramidoyl chloridate prodrug moiety 19 was then coupled to provide 4a in 21% yield, as an ∼1:1 diastereomeric mixture. (23) The two diastereomers were resolved using chiral HPLC to afford the Sp isomer 4b and Rp isomer 4c, respectively. (38) While this route initially provided quantities of 4b, the variability in yields, suboptimal selectivity, frequent use of cryogenic temperatures, and chiral chromatography hindered this route from being suited to larger scales.
The second generation route enabled the diastereoselective synthesis of the single Sp isomer 4b on scales suitable to advance the compound into preclinical efficacy and toxicity studies (Scheme 2). (17) The glycosylation step employed the iodo-base 20 instead of the bromo base, which enabled a more facile metal–halogen exchange compatible with i-PrMgCl·LiCl complex. (39) Treatment with PhMgCl and TMSCl provided 6N protection to remove the acidic protons with a higher degree of control, and addition of i-PrMgCl·LiCl followed by the ribolactone 14 at −20 °C afforded the glycosylation product 16 in a 40% yield. The milder reagents and temperature enabled large-scale batches to be carried out with consistent yields. Treatment of 16 with TMSCN, TMSOTf, and TfOH at −78 °C afforded 17 in 85% yield in >95:5 anomeric ratio. The inclusion of TfOH was key to promote the high yield and high selectivity favoring the desired β-anomer. Benzyl deprotection was effected through treatment with BCl3, and 4 was readily isolated through crystallization. Acetonide protection of the 2′,3′-hydroxyl moieties with 2,2-dimethoxypropane in the presence of H2SO4 afforded 21 in 90% yield. Utilizing the 2′,3′-acetonide protection was found to be optimal as the yield of the coupling reaction with the p-nitrophenolate 2-ethylbutyl-l-alaninate prodrug mixture 22a was dramatically improved compared to directly coupling to the unprotected nucleoside 4 (70% vs 40%). In the event, reaction of 21 with the single Sp isomer of the p-nitrophenolate prodrug precursor 22b in the presence of MgCl2 and Hünig’s base efficiently appended the prodrug group in 70% yield as a single Sp isomer. Final deprotection of the acetonide with concentrated HCl in THF afforded 4b in 69% yield.
Scheme 2
Scheme 2. Second Generation Synthesis of 4ba
aReagents and conditions: (a) TMSCl, PhMgCl, i-PrMgCl·LiCl, THF, – 20 °C, 40%; (b) TMSCN, TfOH, TMSOTf, CH2Cl2, – 78 °C, 85%; (c) BCl3, CH2Cl2, – 20 °C, 86%; (d) 2,2-dimethoxypropane, H2SO4, acetone, rt, 90%; (e) 22b, MgCl2, (i-Pr)2NEt, MeCN, 50 °C, 70%; (f) 37% HCl, THF, rt, 69%; (g) OP(OPh)Cl2, Et3N, CH2Cl2, – 78 °C, then 4-nitrophenol, Et3N, 0 °C, 80%; (h) i-Pr2O, 39%.
The single Sp isomer 22b of the p-nitrophenolate 2-ethylbutyl-l-alaninate prodrug precursor 22a was prepared through a sequence beginning with exposure of 2-ethylbutyl-l-alanine 18a to OP(OPh)Cl2, followed by 4-nitrophenol, to afford 22a as a diastereomeric mixture at phosphorus. Importantly, the single Sp isomer 22b was readily resolved from the mixture in 39% yield through crystallization in diisopropyl ether, a discovery that was paramount for the success of the diastereoselective synthesis of the 4b. (40) Thus, utilizing the p-nitrophenolate 2-ethylbutyl-l-alaninate prodrug coupling partner 22b offered a significant advantage over the chloridate 19 in the first generation sequence. Overall the second generation synthesis of 4b offered the following improvements: (1) milder glycosylation conditions at higher temperature to allow for consistent yields and scalability, (2) higher selectivity and yield for the 1′-cyanation reaction, and (3) a highly efficient coupling sequence of a single Sp prodrug moiety for the diastereoselective synthesis of 4b. Through this second generation route >200 g was rapidly prepared to support preclinical efficacy and toxicity studies.
The stereochemistry of the p-nitrophenolate 2-ethylbutyl-l-alaninate prodrug 22b and candidate compound 4b were unambiguously assigned by small molecule X-ray crystallography (Figure 3). In both cases the Sp isomer was established and suggests that the coupling with the nucleoside and reagent follows a SN2 type inversion of the phosphorus stereocenter.
Figure 3
Figure 3. Thermal ellipsoid representations of (a) 22b and (b) 4b.
The improved method for preparing 4 enabled many monophosphoramidate and bisphosphoramide prodrug analogues to be synthesized, the results of which are summarized in Scheme 3. A number of conditions were identified to affect the coupling of the prodrug moieties to 4 or the 2′,3′-acetonide protected analogue 21. The reactions employing 4 proceeded under either Brønsted basic conditions utilizing t-BuMgCl or Lewis acidic conditions with MgCl2 in polar aprotic solvents to afford the desired prodrugs in yields ranging from 10 to 43%. The coupling reaction of the 2′,3′-acetonide protected analogue 21 under Lewis acidic conditions followed by in situ acetonide deprotection in general afforded much higher yields ranging from 60 to 70% (analogues 4b and 4n). Both the p-nitrophenol (PNP) and pentafluorophenol (PFP) prodrug electrophiles were compatible in the coupling reactions and typically achieved comparable yields. The reactions utilizing diastereomeric mixtures of the prodrug electrophiles, 22a,d–h,j,l,n provided the prodrug products in 1.5–2.6 to 1 diastereomeric ratios (unassigned) at phosphorus. In addition to the monophosphoramidate prodrugs, two bisphosphoramide prodrugs, 4o and 4p, were synthesized and evaluated since they avoided the preparation of chiral phosphorus reagents.
Scheme 3
Scheme 3. Prodrug Synthesis
aProdrug is an undetermined mixture of diastereoisomers unless otherwise indicated.
baa = amino acid, Ala = alanine, Phe = phenylalanine, AIB = 2-aminoisobutyrate, c-Bu = cyclobutyl, c-Pent = cyclopentyl, Pent = pentyl, Neopent = neopentyl, 2-EtBu = 2-ethylbutyl, PNP = p-nitrophenolate, and PFP = pentafluorophenolate.
cLG = leaving group.
dSingle Sp isomer.
eReagent was a single unassigned isomer at phosphorus.
fNA = not applicable.
The monophosphate prodrugs can improve the potency of the parent nucleosides substantially by delivering the monophosphate into cells and effectively bypassing a rate limiting first phosphorylation step. The phenol and amino acid esters mask the negative charge of the monophosphate group enabling facile passive penetration into the cell. The prodrug breakdown is initiated by intracellular esterases (e.g., carboxy esterase 1 and cathepsin A) that cleave the ester unraveling the carboxylate moiety, which then continues to breakdown to the monophosphate that serves as the prescursor to synthesis of the intracellular NTP. (17)
Prodrugs 4a–p were evaluated toward EBOV in three cell lines and for human plasma stability (Table 3). In general the antiviral activity trends for EBOV across all three cell lines were similar supporting the efficient conversion of these prodrugs across multiple different cell types. A series of ethyl esters with differing amino acids (entries 2–5) established that the phenylalanine and alanine amino acids were the most promising (Table 3). Given the intended route of administration was intravenous, increasing lipophiliity beyond log D ∼ 2 was considered a potential issue due to solubility concerns. Therefore, to improve potency the emphasis was placed on the less lipophilic and more commonly used alanine amino acid, with subsequent modification of the ester. Nonproximally branched esters of alanine with increasing log D ranging from 0.6 to 2.1 (entries 5, 11, and 12) demonstrated increased potency. Esters that contained proximal branching without cyclic motifs e.g. i-Pr, t-Bu, and 3-pentyl (entries 7, 8, and 10, respectively) were generally less active, consistent with the increased steric hindrance that likely slows the cleavage rate by esterases. For example, the proximally branched 3-pentyl ester 4l (entry 10) has comparable log D to that of the neopentyl analogue 4m (entry 11) yet much lower potency. In contrast, cyclic butyl and pentyl esters (entries 6 and 9, respectively) are more potent than 4l despite the proximal branching and lower log D, although the potency in the HeLa cell assay was reduced. The d-Ala 2-EtBu mixture (entry 15) was less potent that the corresponding l-Ala analogue mixture (entry 12), and the two bisphosphoramide prodrugs (entries 16 and 17) also had reduced activity compared to their monoamidate counterparts (entries 5 and 12, respectively). Thus, based on antiviral properties across HeLa and HMVEC cells, the most promising monophosphoramidate prodrugs were the neopentyl ester 4m (single undefined isomer) and 2-EtBu ester mixture 4a. The Sp and Rp isomers of 4a (entries 14 and 13, respectively) were separated and found to be similar in potency, but both were marginally more potent than 4m. For the intended iv route of administration, plasma stability was not deemed critical in the selection process provided sufficient stability (t1/2 > 60 min) was maintained to allow loading of target cells harboring the virus during drug infusion. The selection of 4b was made based on the high potency across multiple cell lines and the crystalline nature of the Sp prodrug reagent 22b that allowed rapid scale up for efficacy and IND enabling studies of the single Sp isomer 4b.
Table 3. Antiviral Activity of Prodrugs 4a–p.a
| entry | compd | ester (R) | aab | EBOV EC50 HeLa (nM) | EBOV EC50 HMVECc (nM) | EBOV EC50 macrod (nM) | CC50 MT4 (μM) | human plasma t1/2 (min) | log D |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 4 | >20000 | 780 | >20000 | >57 | 0.3 | |||
| 2 | 4d | Et | l-Phe | 4380 | 587 | 270 | 15 | 1584 | 1.6 |
| 3 | 4e | Et | l-Val | 7040 | 3151 | >100 | 1584 | 1.2 | |
| 4 | 4f | Et | AIB | 8470 | 1585 | >100 | 1584 | 0.9 | |
| 5 | 4g | Et | l-Ala | 2425 | 636 | 13 | 1584 | 0.6 | |
| 6 | 4h | c-Bu | l-Ala | 420 | 88 | 6 | 815 | 1.1 | |
| 7 | 4i | i-Pr | l-Ala | 1845 | 367 | 297 | 21 | 1561 | 1.1 |
| 8 | 4j | t-Bu | l-Ala | 30410 | 3790 | >100 | 1584 | 1.3 | |
| 9 | 4k | c-Pent | l-Ala | 633 | 160 | 120 | 8.8 | 1578 | 1.3 |
| 10 | 4l | 3-Pent | l-Ala | 1810 | 845 | >100 | 860 | 1.6 | |
| 11 | 4m | Neopent | l-Ala | 168 | 92 | 3 | 700 | 1.7 | |
| 12 | 4a | 2-EtBu | l-Ala | 170 | 121 | 100 | 2 | 195 | 2.1 |
| 13 | 4c | 2-EtBu | l-Ala | 80 | 53 | 111 | 3 | 234 | 2.0 |
| 14 | 4b | 2-EtBu | l-Ala | 100 | 53 | 86 | 1.7 | 69 | 2.1 |
| 15 | 4n | 2-EtBu | d-Ala | 550 | 518 | 729 | 42 | 1584 | 2.1 |
| 16 | 4o | Et | l-Ala | 20420 | 9102 | >53 | <0.3 | ||
| 17 | 4p | 2-EtBu | l-Ala | 970 | 678 | 9 | 507 | 2.7 |
a
Data is at least n ≥ 2 unless otherwise reported.
b
aa = Amino Acid, Ala = Alanine, Phe = Phenylalanine, AIB = 2-aminoisobutyrate, c-Bu = cyclobutyl, c-Pent = cyclopentyl, Pent = pentyl, 2-EtBu = 2-ethylbutyl.
c
HMVEC = TERT-immortalized human foreskin microvascular endothelial cells (ATCC-4025).
d
Macro = human macrophages.
In vivo efficacy evaluation of 4b was conducted in monkeys since this represented the most relevant animal model of EVD with similar pathophysiology to the actual human disease. In addition, phosphoramidate esters are highly prone to plasma metabolism in rodents on account of high expression of plasma carboxylesterases, (41) thereby excluding pilot efficacy studies in small animal models. Due to the high first pass hepatic extraction of phosphoramidates, oral administration was also not explored in favor of injectable routes of administration. Moreover, oral delivery in patients acutely infected with EBOV that are demonstrating symptoms of the disease may not be ideal because gastrointestinal symptoms may limit the dose that is effectively absorbed. Intravenous administration of 4b in rhesus monkeys demonstrated rapid elimination of prodrug and appearance of parent nucleoside in systemic circulation (Figure 4a). However, 4b also rapidly distributed to peripheral blood mononuclear cells (PBMCs) and triphosphate levels in PBMCs were elevated to a maximum within 2 h (Figure 4b). A dose of 10 mg/kg resulted in an estimated PBMC triphosphate level at 24 h that was several-fold higher than the estimated IC50 of 5 μM for EBOV, with a half-life of 14 h similar to that measured in vitro in human macrophages. The long intracellular half-life of 4tp supported once-daily iv administration in the rhesus efficacy study and in the clinical program.
Figure 4
Figure 4. Concentration–time profiles following 10 mg/kg iv single dose slow bolus administration of 4b in Rhesus (mean ± SD, n = 3 per time point). (a) Plasma profile of prodrug 4b (black circle) and parent nucleoside 4 (blue triangle). (b) Intracellular concentration of active metabolite 4tp in PBMCs (green diamond) and estimated 4tp EBOV IC50 = 5 μM (dashed black line).
Preclinical in vivo efficacy for 4b was conducted in an EBOV-infected rhesus challenge model. The overall strategy was to determine a maximally efficacious dose of 4b that could be safely administered and to understand whether delayed time of treatment would be effective, a property that was considered critical for the successful clinical application of 4b. These studies have recently been published in full and will only be summarized here. (17) The starting dose of 3 mg/kg iv was modeled based on the 4tp levels in rhesus PBMCs following 10 mg/kg iv dosing to have the potential for inhibiting Ebola viral replication in vivo. A correlation of the in vitro antiviral activity and intracellular metabolism described earlier suggested the 3 mg/kg dose would produce 4tp levels that exceed the estimated IC50 for most of the dosing interval. Compound 4b was dosed iv at 3 mg/kg on day 0 or day 2 relative to EBOV inoculation, and continued once daily for 12 days. Systemic viremia was reduced and survival out to day 28 postinfection was improved. Animals administered 3 mg/kg 4b, starting at day 0, had a survival rate of 33% while those initiated on day 3 had a 66% survival at 28 days. These encouraging data were then followed with a second study in which one arm explored dosing initiation on day 3 with 10 mg/kg daily for 12 days to assess whether increased dose compared to the 3 mg/kg result could be more efficacious. Two other arms explored an initial loading dose of 10 mg/kg on day 2 or day 3, followed by 11 3 mg/kg daily maintenance doses. All of the animals in the two arms in which 4b treatments were initiated on day 3 (n = 12 total) survived through day 28, the end of study. In the daily 10 mg/kg dosed group the effect on viremia was consistently greater than in any of the other groups and was below the limit of quantitation (8 × 104 RNA copies mL–1) in four of the six animals on days 5 and 7 relative to the vehicle-treated control in which the geometric mean exceeded 109 copies mL–1 at these time points. These data established the effectiveness of 4b in treatment of EVD in NHPs and accelerated its progress into clinical development. In addition to the efficacy studies, distribution studies in cynomolgus monkeys using [14C]4b at the same effective dose of 10 mg/kg established the presence of drug-related products in the potential sanctuary sites for the virus including testes, epididymis, eyes, and brain. (17) At 4 h postdose, the drug levels in the testes and epididymis exceeded those observed in plasma, and even at 168 h postdose detectable levels of drug-related products were still observed in the testes. Exposure levels in the brain were lower than other tissues, including plasma at 4 h, but were detectable above the plasma levels at 168 h indicating a long half-life of exposure in brain relative to plasma. These data supported the potential that 4b treatment may also reduce persistence of virus in these sanctuary sites.
Safety and pharmacokinetics of 4b administered as once-daily iv infusion were evaluated in single and multiple dose phase 1 clinical trials. No serious adverse effects of the drug were observed. During the course of the phase 1 studies two requests for a compassionate use of 4b were received. The first case involved a healthcare worker who had survived acute infection but had relapsed with symptoms of acute meningoencephalytis. (42) Ebola virus was detected both systemically in plasma and in cerebrospinal fluid. When treated with monoclonal antibodies, the patient developed adverse reaction and was subsequently treated with supportive therapy and 4b for a period of 14 days beginning with a dose of 150 mg and then increasing to 225 mg after two daily infusions. No serious adverse effects related to drug were observed. The patient recovered and cleared the virus from both plasma and CNS although without proper control or natural history data to compare, it is not clear whether the antiviral therapy was effective. The second case involved a newborn infant congenitally infected with EBOV and treated with monoclonal antibodies, blood transfusion, and subsequently 4b. (43) The infant recovered and was eventually declared free of Ebola virus after repeated testing failed to detect viremia. The unprecedented scale of the West African epidemic and the ability to reduce mortality rates through supportive therapy has resulted in many survivors of EVD. The persistence of virus sequelae in survivors in multiple body compartments has now been documented in addition to secondary sexual transmission via virus in genital secretions. (4) This has prompted the initiation of a randomized, blinded, placebo-controlled phase 2 study (PREVAIL IV) which plans to enroll at least 60 adult male survivors to receive either 100 mg of 4b or placebo once-daily over 5 days to assess the effect of 4b therapy on the viral shedding in semen. (44) The results of this study could provide the first evidence as to the potential of 4b to reduce virus replication in humans.
Conclusion
Click to copy section linkSection link copied!
The recent EBOV outbreak prompted the urgent need for antiviral therapeutics for the treatment of EVD. We identified a promising nucleotide therapeutic 4b through initial screening and subsequent optimization of the prodrug moiety for iv administration. The partnership with government organizations, including CDC and USAMRIID, that generated the screening data and conducted the rhesus efficacy studies was critical to the successful identification of 4b. Also of importance was the significant chemistry effort that rapidly identified a more efficient route to the parent compound 4 and the ability to prepare, through crystallization of a key reagent, the single Sp phosphorus diastereoisomer 4b for in vivo model studies. The active triphosphate delivered by the prodrug has low micromolar polymerase activity toward EBOV, high selectivity for the viral polymerase compared to host polymerases, and a long intracellular half-life supporting once-daily administration. Parenteral treatment with 4b in EBOV infected NHPs at 10 mg/kg over 12 days demonstrated a substantial antiviral effect along with 100% survival. Based on its promising potential, and preliminary safety data from phase 1 studies, regulatory authorities approved the compassionate use of 4b in two cases including an newborn infant with EVD. Further clinical data on 4b is being collected in the phase 2 PREVAIL IV study that aims to assess the ability of 4b to reduce persistence of EBOV in sanctuary sites of survivors.
Experimental Section
Click to copy section linkSection link copied!
All organic compounds were synthesized at Gilead Sciences, Inc. (Foster City, CA, USA) unless otherwise noted. Commercially available solvents and reagents were used as received without further purification. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury Plus 400 MHz instrument at room temperature, with tetramethylsilane as an internal standard. Proton nuclear magnetic resonance spectra are reported in parts per million (ppm) on the δ scale and are referenced from the residual protium in the NMR solvent (chloroform-d1, δ 7.26; methanol-d4, δ 3.31; DMSO-d6, δ 2.50). Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sep = septet, m = multiplet, br = broad, app = apparent); coupling constants (J) in hertz; integration. Carbon-13 nuclear magnetic resonance spectra are reported in parts per million on the δ scale and are referenced from the carbon resonances of the solvent (chloroform-d1, δ 77.16, methanol-d4, δ 49.15; DMSO-d6, δ 39.52). Data are reported as follows: chemical shift. No special nomenclature is used for equivalent carbons. Phosphorus-31 nuclear magnetic resonance spectra are reported in parts per million on the δ scale. Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet); coupling constants (J) in hertz. No special nomenclature is used for equivalent phosphorus resonances. Analytical thin-layer chromatography was performed using Merck KGaA silica gel 60 F254 glass plates with UV visualization. Preparative normal phase silica gel chromatography was carried out using a Teledyne ISCO CombiFlash Companion instrument with silica gel cartridges. Purities of the final compounds were determined by high-performance liquid chromatography (HPLC) and were greater than 95% unless otherwise noted. HPLC conditions to assess purity were as follows: Agilent 1100 Series HPLC, Phenominex Kinetex C18; 2.6 μm,100 Å, 100 × 4.6 mm2 column, 2–98% gradient of 0.1% trifluoroacetic acid in water, and 0.1% trifluoroacetic acid in acetonitrile; flow rate, 1.5 mL/min; acquisition time, 8.5 min; wavelength, UV 214 and 254 nm. High-resolution mass spectrometry (HRMS) was performed on an Agilent model 6230 accurate mass time of flight mass spectrometer featuring Agilent Jet Stream Thermal Focusing Technology, with an Agilent 1200 Rapid Resolution HPLC. HRMS chromatography was performed using an Agilent Zorbax Eclipse Plus C18 RRHD 1.8 μm, 2.1 × 50 mm2 column at 30 °C, with a 10–90% gradient of 0.05% trifluoroacetic acid in water and 0.05% trifluoroacetic acid in acetonitrile. LC-MS (MS) was conducted on a Thermo Finnigan MSQ Std. using electrospray positive and negative [M + 1]+ and [M – 1]−, and a Dionex Summit HPLC System (model P680A HPG) equipped with a Gemini 5 μ C18 110A column (30 mm × 4.60 mm), eluting with 0.05% formic acid in 1% acetonitrile/water and 0.05% formic acid in 99% acetonitrile/water. Optical rotations were recorded on a Jasco P-2000 polarimeter.
The synthesis, characterization data, and associated references for the following compounds are provided in the Supporting Information: 4, 4a, 7–11, 9a, 11a, 12–13, 13a, 13b, 4tp, 13tp, 16–17, 18a, 19, 21, and 22a–n.
(S)-2-Ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4b)
Compound 4b was prepared from 4 and 22b as described previously. (17)1H NMR (400 MHz, methanol-d4): δ 7.86 (s, 1H), 7.33–7.26 (m, 2H), 7.21–7.12 (m, 3H), 6.91 (d, J = 4.6 Hz, 1H), 6.87 (d, J = 4.6 Hz, 1H), 4.79 (d, J = 5.4 Hz, 1H), 4.43–4.34 (m, 2H), 4.28 (ddd, J = 10.3, 5.9, 4.2 Hz, 1H), 4.17 (t, J = 5.6 Hz, 1H), 4.02 (dd, J = 10.9, 5.8 Hz, 1H), 3.96–3.85 (m, 2H), 1.49–1.41 (m, 1H), 1.35–1.27 (m, 8H), 0.85 (t, J = 7.4 Hz, 6H). 13C NMR (100 MHz, methanol-d4): δ 174.98, 174.92, 157.18, 152.14, 152.07, 148.27, 130.68, 126.04, 125.51, 121.33, 121.28, 117.90, 117.58, 112.29, 102.60, 84.31, 84.22, 81.26, 75.63, 71.63, 68.10, 67.17, 67.12, 51.46, 41.65, 24.19, 20.56, 20.50, 11.33, 11.28. 31P NMR (162 MHz, methanol-d4): δ 3.66 (s). HRMS (m/z): [M]+ calcd for C27H35N6O8P, 602.2254; found, 602.2274. [α]21D – 21 (c 1.0, MeOH).
(S)-2-Ethylbutyl 2-(((R)-(((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4c)
Compound 4c was prepared from 4a by chiral chromatography. Compound 4a was dissolved in acetonitrile. The resulting solution was loaded onto a Lux Cellulose-2 chiral column, equilibrated in acetonitrile, and eluted with isocratic acetonitrile/methanol (95:5 (v/v)). The first eluting compound was the Rp diastereomer 4c, and the second eluting compound was the Sp diastereomer 4b.1H NMR (400 MHz, methanol-d4): δ 8.05 (s, 1H), 7.36 (d, J = 4.8 Hz, 1H), 7.29 (br t, J = 7.8 Hz, 2H), 7.19–7.13 (m, 3H), 7.11 (d, J = 4.8 Hz, 1H), 4.73 (d, J = 5.2 Hz, 1H), 4.48–4.38 (m, 2H), 4.37–4.28 (m, 1H), 4.17 (t, J = 5.6 Hz, 1H), 4.08–3.94 (m, 2H), 3.94–3.80 (m, 1H), 1.48 (sep, J = 12.0, 6.1 Hz, 1H), 1.34 (p, J = 7.3 Hz, 4H), 1.29 (d, J = 7.2 Hz, 3H), 0.87 (t, J = 7.4 Hz, 6H). 31P NMR (162 MHz, methanol-d4): δ 3.71 (s). MS m/z: 603.1 [M + 1]. [α]21D – 46 (c 1.0, MeOH).
(2S)-Ethyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (4d)
Compound 4 (0.030 g, 0.103 mmol) was dissolved in DMF (1 mL), and then THF (0.5 mL) was added. t-BuMgCl (1 M/THF, 154.5 μL, 0.154 μmol) was added to the reaction in a dropwise manner with vigorous stirring. The resulting white slurry was stirred at rt for about 30 min. A solution of compound 22d (0.058 g, 0.124 mmol) in THF (1 mL) was added in a dropwise manner to the reaction at rt. The reaction progress was monitored by LC-MS. When the reaction progressed to 50% conversion, the reaction was cooled in an ice bath and quenched with glacial acetic acid (70 μL). The reaction was concentrated and the crude residue was purified by reverse phase preparatory HPLC to afford compound 4d (22 mg, 34%, as a 2.6:1 mixture of diastereomers at phosphorus). 1H NMR (400 MHz, DMSO-d6): δ 7.91 (d, J = 4 Hz, 1H), 7.90 (br s, 2H), 7.09–7.30 (m, 8H), 7.01, (t, J = 8.2 Hz, 2H), 6.89 (d, J = 4.4 Hz, 1H), 6.82 (t, J = 4.4 Hz, 1H), 6.27 (m, 1H), 6.14 (m, 1H), 5.34 (m, 1H), 4.62 (t, J = 5.6 Hz, 1H), 4.15 (m, 1H), 4.01−3.78 (m, 6H), 2.92 (m, 1H), 2.78 (m, 1H), 1.04 (m, 3H). 31P NMR (162 MHz, DMSO-d6) δ 3.69 (s), 3.34 (s). MS m/z = 623.0 [M+1].
(2S)-Ethyl 2-(((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoate (4e)
Compound 4 (0.040 g, 0.14 mmol) was dissolved in NMP (1.5 mL) and then THF (0.25 mL) was added. This solution was cooled in an ice bath and t-BuMgCl (1M/THF, 425.7 μL, 0.426 μmol) was added in a dropwise manner with vigorous stirring. The ice bath was removed, and the resulting white slurry was stirred at rt for about 15 min. A solution of compound 22e (0.081 g, 0.192 mmol) in THF (0.5 mL) was added in a dropwise manner to the reaction at rt. The reaction progress was monitored by LC-MS. When the reaction progressed to 50% conversion, the reaction was cooled in an ice bath and quenched with glacial acetic acid (70 μL). The reaction was concentrated and crude residue was semipurified from the residue by reverse phase HPLC. The semipure material was further purified by silica gel column chromatography (eluent, 100% EtOAc ramping to 10% MeOH in EtOAc) to afford compound 4e (0.034 g, 43% as a 1.8:1 mixture of diastereomers). 1H NMR (400 MHz, DMSO-d6): δ 7.91 (d, J = 1.6 Hz, 1H), 7.88 (br s, 2H), 7.32 (m, 2H), 7.15 (m, 3H), 6.90 (t, J = 4.2 Hz, 1H), 6.84 (d, J = 4.8 Hz, 1H), 6.26 (dd, J = 13.4, 6.2 Hz, 1H), 5.87 (q, J = 11.2 Hz, 1H), 5.35 (m, 1H), 4.64 (m, 1H), 4.25 (m, 2H), 4.15−3.93 (m, 4H), 3.45 (m, 1H), 1.87 (m, 1H), 1.16−1.09 (m, 3H), 0.83−0.70 (m, 6H). 31P NMR (162 MHz, DMSO-d6): δ 4.59 (s), 4.47 (s). MS m/z = 575.02 [M + 1].
Ethyl 2-(((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate (4f)
Compound 4 (66 mg, 0.23 mmol) was dissolved in NMP (2.0 mL) and the mixture was cooled to about 0 °C. t-BuMgCl (1.0 M in THF, 0.34 mL, 0.34 mmol) was then added and the resulting mixture was stirred at 0 °C for about 30 min. A solution of compound 22f (139 mg, 0.34 mmol) in THF (1.0 mL) was then added, and the reaction mixture was heated to about 50 °C. After about 2 h, the reaction was cooled to rt and quenched with acetic acid and methanol. The resulting mixture was concentrated under reduced pressure and purified by preparatory reverse phase HPLC to afford compound 4f (32 mg, 25% as a 1.5:1 mixture of diastereomers). 1H NMR (400 MHz, DMSO-d6): δ 7.89 (m, 3H), 7.31 (q, J = 8.1 Hz, 2H), 7.22–7.05 (m, 3H), 6.87 (d, J = 4.5, 1H), 6.80 (d, J = 4.5 Hz, 1H), 6.27 (d, J = 11.7, 1H), 5.81 (d, J = 9.7, 1H), 5.35 (d, J = 5.6 Hz, 1H), 4.64 (dt, J = 9.0, 5.6 Hz, 1H), 4.24 (m, 2H), 4.11 (m, 1H), 4.04–3.90 (m, 3H), 1.39–1.23 (m, 6H), 1.10 (t, J = 7.1, 3H). 31P NMR (162 MHz, DMSO-d6): δ 2.45, 2.41. MS m/z = 561.03 [M + 1].
Ethyl ((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-l-alaninate (4g)
Compound 4 (50 mg, 0.17 mmol) was dissolved in NMP–THF (1:1 mL)) and cooled with an ice bath. t-BuMgCl (1.0 M in THF, 0.257 mL, 0.257 mmol) was then added over about 5 min. The resulting mixture was allowed to warm to rt and was stirred for about 30 min. Then a solution of compound 22g (74.6 mg, 0.189 mmol) in THF (2 mL) was added. After about 30 min, the reaction mixture was purified by preparatory reverse phase HPLC. Fractions containing the desired product were further purified with silica gel chromatography (eluent: 0–20% methanol in dichloromethane) to afford compound 4g (23 mg, 24% as a 2.5:1 mixture of diastereomers). 1H NMR (400 MHz, methanol-d4): δ 7.76 (d, J = 6.0 Hz, 1H), 7.25–7.14 (m, 2H), 7.11–6.99 (m, 3H), 6.87–6.72 (m, 2H), 4.70 (d, J = 5.4 Hz, 1H), 4.39–4.24 (m, 2H), 4.20 (dddd, J = 9.7, 7.9, 5.1, 2.8 Hz, 1H), 4.10 (dt, J = 12.8, 5.5 Hz, 1H), 4.06–3.91 (m, 2H), 3.72 (ddq, J = 14.3, 9.3, 7.1 Hz, 1H), 1.17 (dd, J = 7.1, 1.0 Hz, 1H), 1.14–1.06 (m, 5H). 31P NMR (162 MHz, methanol-d4): δ 3.73, 3.68. MS m/z = 547 [M + 1].
(2S)-Cyclobutyl 2-(((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4h)
Compound 4 (58 mg, 0.20 mmol) was mixed with compound 22h (101 mg, 0.240 mmol) in 2 mL of anhydrous DMF. Magnesium chloride (42 mg, 0.44 mmol) was added in one portion. The reaction mixture was heated to about 50 °C. N,N-Diisopropylethylamine (87 μL, 0.5 mmol) was added, and the reaction was stirred for about 2 h at about 50 °C. The reaction mixture was cooled to room temperature, was diluted with ethyl acetate, and was washed with 5% aqueous citric acid solution followed by saturated aqueous sodium chloride solution. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified with silica gel column (eluent, 0–5% methanol in dichloromethane) to afford compound 4h (42 mg, 37% yield, as a 3:2 mixture of diastereomers). 1H NMR (400 MHz, methanol-d4): δ 7.85 (m, 1H), 7.34–7.22 (m, 2H), 7.22–7.08 (m, 3H), 6.94–6.84 (m, 2H), 4.95–4.85 (m, 1H), 4.79 (m, 1H), 4.46–4.34 (m, 2H), 4.34–4.24 (m, 1H), 4.19 (m, 1H), 3.81 (m, 1H), 2.27 (m, 2H), 2.01 (m, 2H), 1.84–1.68 (m, 1H), 1.62 (m, 1H), 1.30–1.16 (m, 3H). 31P NMR (162 MHz, methanol-d4): δ 3.70, 3.65. MS m/z = 573.0 [M + 1].
(S)-Isopropyl 2-(((R)-(((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4i)
Compound 4 (60.0 mg, 206 μmol) was dissolved in NMP (0.28 mL). THF (0.2 mL) was added followed by tert-butyl magnesium chloride (1.0 M solution in tetrahydrofuran, 0.309 mL) at rt under an argon atmosphere. After 20 min, a solution of compound 22i (81 mg, 206 μmol) in THF (0.2 mL) was added, and the resulting mixture was warmed to about 50 °C. After 3 h, the reaction mixture was allowed to cool to rt and was purified directly by preparatory HPLC to afford compound 4i (44 mg, 38% as a single diastereomer). 1H NMR (400 MHz, methanol-d4): δ 7.86 (s, 1H), 7.34–7.26 (m, 2H), 7.21–7.12 (m, 3H), 6.91 (d, J = 4.6 Hz, 1H), 6.87 (d, J = 4.6 Hz, 1H), 4.92 (sep, J = 6.3 Hz, 1H), 4.80 (d, J = 5.4 Hz, 1H), 4.43–4.34 (m, 1H), 4.33–4.24 (m, 1H), 4.18 (t, J = 5.6 Hz, 1H), 3.82 (dq, J = 9.7, 7.1 Hz, 2H), 1.27 (dd, J = 7.1, 1.0 Hz, 3H), 1.18 (dd, J = 6.3, 4.8 Hz, 6H). 31P NMR (162 MHz, methanol-d4): δ 3.72 (s). MS m/z = 561.11 [M + 1].
tert-Butyl ((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-l-alaninate (4j)
To a mixture of intermediate 4 (80 mg, 0.28 mmol), intermediate 22j (174 mg, 0.41 mmol), and MgCl2 (39 mg, 0.41 mmol) in DMF (4 mL) was added N,N-diisopropylethylamine (0.12 mL, 0.69 mmol) dropwise at room temperature. The reaction mixture was stirred at 50 °C for 1 h and was cooled to rt. The resulting mixture was concentrated under reduced pressure to approximately 2 mL volume and was purified by reverse phase preparative HPLC. Fractions containing the desired product were combined and further purified by silica gel column chromatography (eluent, 0–20% methanol in methylene chloride) to afford compound 4j (51 mg, 32%, 1.5:1 diastereomeric mixture). 1H NMR (400 MHz, methanol-d4): δ 7.86 (s, 0.4H), 7.84 (s, 0.6H), 7.28 (m, 2H), 7.21–7.10 (m, 3H), 6.96–6.83 (m, 2H), 4.79 (m, 1H), 4.46–4.34 (m, 2H), 4.28 (m, 1H), 4.22–4.13 (m, 1H), 3.81–3.64 (m, 1H), 1.40 (m, 9H), 1.22 (m, 3H). 31P NMR (162 MHz, methanol-d4): δ 3.79 (s). MS m/z = 575 [M + 1].
(2S)-Cyclopentyl 2-(((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4k)
Compound 4 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooled under an ice water bath. Then 1 M t-BuMgCl (0.52 mL, 0.77 mmol) was added dropwise slowly. The resulting mixture was stirred for about 30 min at rt. Then compound 22k (247 mg, 0.52 mmol) in THF (2 mL) was added over about 5 min and the resulting mixture was stirred for about 24 h at rt. The resulting mixture was diluted with ethyl acetate, cooled under ice–water bath, treated with aqueous NaHCO3 (2 mL), washed with brine, dried with sodium sulfate, and concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography (eluent, 0–20% methanol in dichloromethane) followed by reverse phase preparatory HPLC to afford compound 4k (47 mg, 23% as a 27:1 mixture of diastereomers). 1H NMR (400 MHz, methanol-d4): δ 7.85 (s, 1H), 7.33–7.22 (m, 2H), 7.14 (tdd, J = 7.6, 2.1, 1.1 Hz, 3H), 6.95–6.87 (m, 2H), 5.13–5.00 (m, 1H), 4.78 (d, J = 5.4 Hz, 1H), 4.48–4.35 (m, 2H), 4.30 (ddd, J = 10.6, 5.7, 3.6 Hz, 1H), 4.19 (t, J = 5.4 Hz, 1H), 3.78 (dq, J = 9.2, 7.1 Hz, 1H), 1.81 (dtd, J = 12.5, 5.9, 2.4 Hz, 2H), 1.74–1.49 (m, 6H), 1.21 (dd, J = 7.1, 1.2 Hz, 3H). MS m/z = 587 [M + 1].
Pentan-3-yl ((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-l-alaninate (4l)
To a mixture of compound 4 (80 mg, 0.28 mmol), 22l (170 mg, 0.39 mmol), and MgCl2 (39 mg, 0.41 mmol) in DMF (4 mL) was added N,N-diisopropylethylamine (0.12 mL, 0.69 mmol) dropwise at rt. The resulting mixture was stirred at 50 °C for 1 h, concentrated to approximately 2 mL volume, and purified by reverse phase preparative HPLC to afford compound 4l (68 mg, 42%, 1.5:1 diastereomeric mixture). 1H NMR (400 MHz, methanol-d4): δ 7.86 (s, 0.4 H), 7.85 (s, 0.6 H), 7.33–7.23 (m, 2H), 7.21–7.08 (m, 3H), 6.95–6.84 (m, 2H), 4.79 (m, 1H), 4.69 (m, 1H), 4.47–4.34 (m, 2H), 4.34–4.24 (m, 1H), 4.19 (m, 1H), 3.85 (m, 1H), 1.64–1.42 (m, 4H), 1.29 (dd, J = 7.0, 1.1 Hz, 1.1 H), 1.23 (dd, J = 7.2, 1.3 Hz, 1.9H), 0.91–0.76 (m, 6H). 31P NMR (162 MHz, methanol-d4): δ 3.71, 3.69. MS m/z = 589 [M + 1].
(S)-Neopentyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4m)
Compound 4 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooled under ice water bath. Then 1 M t-BuMgCl (0.52 mL, 0.77 mmol) was added dropwise slowly. The resulting mixture was stirred for 30 min at room temperature. Then compound 22m (248 mg, 0.52 mmol) was added over 5 min, and the resulting mixture was stirred for 24h at room temperature, diluted with EtOAc, cooled under ice–water bath, treated with aqueous NaHCO3 (2 mL), washed with brine, dried with sodium sulfate, and concentrated in vacuo. The resulting mixture was purified by silica gel column chromatography (MeOH 0 to 20% in DCM) and prep-HPLC (acetonitrile 10 to 80% in water) to give compound 4m (12 mg, 10% as a single diastereomer). 1H NMR (400 MHz, methanol-d4): δ 7.86 (s, 1H), 7.36–7.24 (m, 2H), 7.23–7.10 (m, 3H), 6.96–6.85 (m, 2H), 4.78 (d, J = 5.4 Hz, 1H), 4.38 (tdd, J = 10.0, 4.9, 2.5 Hz, 2H), 4.32–4.24 (m, 1H), 4.17 (t, J = 5.6 Hz, 1H), 3.91 (dq, J = 9.8, 7.1 Hz, 1H), 3.81 (d, J = 10.5 Hz, 1H), 3.69 (d, J = 10.5 Hz, 1H), 1.31 (dd, J = 7.2, 1.1 Hz, 3H), 0.89 (s, 9H). MS m/z = 589 [M + 1]+
2-Ethylbutyl ((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-d-alaninate (4n)
Compound 21 (50 mg, 0.15 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) and stirred under atmospheric argon. Compound 22n (75 mg, 0.17 mmol) was added followed by magnesium chloride (21 mg, 0.23 mmol), and the reaction was warmed to 50 °C and stirred for 30 min. N,N-diisopropylethylamine (65.0 μL, 0.375 mmol) was added dropwise, and the reaction mixture was stirred for 3 h at 50 °C. The reaction mixture was then cooled in an ice bath, and 12 N HCl(aq) (175 μL) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at rt for 4 h. The reaction mixture was diluted with ethyl acetate (15 mL) and cooled in an ice bath. Aqueous 1 N NaOH solution was added slowly to give pH of 10. The organic layer was then washed with 5% aqueous sodium carbonate solution and then saturated aqueous sodium chloride solution. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified with silica gel column chromatography (eluent, 0–10% methanol in dichloromethane) to afford compound 4n (60 mg, 66% yield as a 1.1:1 diastereomeric mixture). 1H NMR (400 MHz, methanol-d4): δ 7.87–7.83 (m, 1H), 7.37–7.22 (m, 2H), 7.22–7.04 (m, 3H), 6.96–6.79 (m, 2H), 4.82–4.75 (m, 1H), 4.45–4.23 (m, 3H), 4.18 (m, 1H), 4.06–3.85 (m, 3H), 1.52–1.38 (m, 1H), 1.38–1.24 (m, 7H), 0.85 (m, 6H). 31P NMR (162 MHz, methanol-d4): δ 3.87, 3.55. MS m/z = 603.1 [M + 1].
(2S,2′S)-Diethyl 2,2′-((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate (4o)
Compound 4 (14.6 mg, 0.05 mmol) was dissolved in anhydrous trimethyl phosphate (0.5 mL) and stirred under N2(g) at rt. POCl3 (9.2 μL, 0.1 mmol) was added, and the mixture stirred for about 60 min. Alanine ethyl ester hydrochloride 18b (61 mg, 0.4 mmol, Aldrich, CAS No. 1115-59-9), and then Et3N (70 μL, 0.5 mmol) was added. The resultant mixture was stirred for about 15 min, and then additional Et3N (70 μL, 0.5 mmol) was added to give a solution pH of 9–10. The mixture was stirred for about 2 h and then diluted with ethyl acetate, washed with saturated aqueous NaHCO3 solution, followed by saturated aqueous NaCl solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC to afford compound 4o (5.5 mg, 16%). 1H NMR (400 MHz, methanol-d4): δ 8.13 (s, 1H), 7.41 (d, J = 4.8 Hz, 1H), 7.18 (d, J = 4.8 Hz, 1H), 4.78 (d, J = 5.6 Hz, 1H), 4.36 (m, 1H), 4.25–4.08 (m, 7H), 3.83 (m, 2H), 1.33–1.23 (m, 12H). 31P NMR (162 MHz, methanol-d4): δ 13.8. MS m/z = 570.0 [M + 1].
(2S,2′S)-Bis(2-ethylbutyl) 2,2′-((((2R,3S,4R,5R)-5-(4-Aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate (4p)
To a suspension of compound 4 (52 mg, 0.18 mmol) and solid sodium bicarbonate (53 mg) in trimethyl phosphate (1.5 mL) at 0 °C was added POCl3 (120 mg, 0.783 mmol). The mixture was stirred at 0 °C for 3 h, at which point a solution of 18a (790 mg, 4.56 mmol) in MeCN (1 mL) was then added. The reaction mixture was stirred at 0 °C for 0.5 h, then triethylamine (0.1 mL) was added and stirred at rt for 0.5 h. The reaction mixture was diluted with ethyl acetate (10 mL), washed with water (10 mL), and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent, 50–100% ethyl acetate in hexanes gradient followed by 0–10% methanol in ethyl acetate gradient) to afford compound 4p (71 mg, 58%). 1H NMR (400 MHz, methanol-d4): δ 7.88 (s, 1H), 6.95 (d, J = 4.8 Hz, 1H), 6.89 (d, J = 4.4 Hz, 1H), 4.85 (d, J = 5.6 Hz, 1H), 4.35−4.32 (m, 1H), 4.26−4.12 (m, 3H), 4.09−4.04 (m, 2H), 3.98−3.94 (m, 2H), 3.89−3.79 (m, 2H), 1.54−1.44 (m, 2H), 1.39−1.27 (m, 14H), 0.89 (t, J = 7.2 Hz, 12H). 31P NMR (162 MHz, methanol-d4): δ 13.83. MS m/z = 682.1 [M + 1].
Supporting Information
Click to copy section linkSection link copied!
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01594.
Homology model of HIV (1RTD (32b)) X-ray structure used to generate the EBOV model for 4tp (PDB)
Homology model of HCV (4WTG (32c)) X-ray structure used to generate the EBOV model for 13tp (PDB)
Molecular formula strings (CSV)
Assay methods, molecular modeling with RSV, Marburg and Sudan viruses, compound synthesis, and single crystal X-ray structure information(PDF)
The Cambridge Crystallographic Data Center (CCDC) numbers for the X-ray structures of compound 22b and 4b are 1445315 and 1525480, respectively.
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
Click to copy section linkSection link copied!
- Richard L. Mackman - Gilead Sciences, Inc., Foster City, California 94404, United States;
http://orcid.org/0000-0001-8861-7205;
Acknowledgments
Click to copy section linkSection link copied!
We acknowledge the contributions of the following individuals at USAMRIID for participation: in vivo studies, J. Wells, K. Stuthman, N. Lackemeyer, S. Van Tongeren, G. Donnelly, J. Steffens, A. Shurtleff, L. Gomba, and J. Benko; scientific input, L. Welch, T. Bocan, A. Duplantier, R. Panchal, C. Kane, and D. Mayers (currently of Cocrystal Pharma, Inc.); and services performed, S Tritsch, C. Retterer, D. Gharaibeh, T. Kenny, B. Eaton, G. Gomba, J. Nuss, and C. Rice. From Gilead Sciences we acknowledge K. Wang, K. Brendza, T. Alfredson, and L. Serafini who assisted with analytical methods, S. Bondy and R. Seemayer procured key raw materials, and L. Heumann, R. Polniaszeck, E. Rueden, A. ChtChemelinine, K. Brak, and B. Hoang contributed to synthesis, Yelena Zherebina to chiral separations, and D. Babusis for DMPK sample analysis data. We also thank Curtis E. Moore from the Rheingold Laboratory, UC San Diego, for assistance in the solving of crystal structures. These studies were in part supported by The Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD) of the Defense Threat Reduction Agency (DTRA) under Plan No. CB10218. CDC core funding supported the work done by M.K.L. at CDC.
| BSL-4 | biosafety level 4 |
| EBOV | Ebola virus |
| EVD | Ebola virus disease |
| HEp-2 | human epithelial type 2 cell |
| HMVEC-TERT | human foreskin microvascular endothelial cells |
| Huh-7 | hepatocellular carcinoma cell |
| IACUC | Institutional Animal Care and Use Committee |
| IND | investigational new drug |
| LG | leaving group |
| log D | logarithm of distribution coefficient |
| Macro | human macrophage cells |
| MERS | Middle East respiratory syndrome |
| MT4 | human leukemia T-cell |
| NHP | non-human primate |
| NMI | N-methylimidazole |
| NTP | nucleoside triphosphate |
| PBMC | peripheral blood mononuclear cell |
| PFP | pentafluorophenol |
| PNP | p-nitrophenol |
| Pol | polymerase |
| POLRMT | mitochondrial RNA polymerase |
| RSV | respiratory syncytial virus |
| SARS | severe acute respiratory syndrome |
| SD | standard deviation |
| SNI | single nucleotide incorporation |
| USAMRIID | United States Army Medical Research Institute of Infectious Diseases |
References
Click to copy section linkSection link copied!
This article references 44 other publications.
- 1World Health Organization. Ebola Situation Report—10 June 2016. http://apps.who.int/iris/bitstream/10665/208883/1/ebolasitrep_10Jun2016_eng.pdf?ua=1 (accessed Jul. 22, 2016).Google ScholarThere is no corresponding record for this reference.
- 2World Health Organization. Ebola Data and Statistics—11 May 2016. http://apps.who.int/gho/data/view.ebola-sitrep.ebola-summary-20160511?lang=en (accessed Jul. 22, 2016).Google ScholarThere is no corresponding record for this reference.
- 3Vetter, P.; Fischer, W. A., II; Schibler, M.; Jacobs, M.; Bausch, D. G.; Kaiser, L. Ebola Virus Shedding and Transmission: Review of Current Evidence. J. Infect. Dis. 2016, 214, S177– S184, DOI: 10.1093/infdis/jiw254Google Scholar3Ebola virus shedding and transmission: review of current evidenceVetter, Pauline; Fischer, William A., II; Schibler, Manuel; Jacobs, Michael; Bausch, Daniel G.; Kaiser, LaurentJournal of Infectious Diseases (2016), 214 (Suppl. 3), S177-S184CODEN: JIDIAQ; ISSN:0022-1899. (Oxford University Press)Background. The magnitude of the 2013-2016 Ebola virus disease outbreak in West Africa was unprecedented, with >28 500 reported cases and >11 000 deaths. Understanding the key elements of Ebola virus transmission is necessary to implement adequate infection prevention and control measures to protect healthcare workers and halt transmission in the community. Methods. We performed an extensive PubMed literature review encompassing the period from discovery of Ebola virus, in 1976, until 1 June 2016 to evaluate the evidence on modes of Ebola virus shedding and transmission. Findings. Ebola virus has been isolated by cell culture from blood, saliva, urine, aq. humor, semen, and breast milk from infected or convalescent patients. Ebola virus RNA has been noted in the following body fluids days or months after onset of illness: saliva (22 days), conjunctiva/tears (28 days), stool (29 days), vaginal fluid (33 days), sweat (44 days), urine (64 days), amniotic fluid (38 days), aq. humor (101 days), cerebrospinal fluid (9 mo), breast milk (16 mo [preliminary data[), and semen (18 mo). Nevertheless, the only documented cases of secondary transmission from recovered patients have been through sexual transmission. We did not find strong evidence supporting respiratory or fomite-assocd. transmission.
- 4Mate, S. E.; Kugelman, J. R.; Nyenswah, T. G.; Ladner, J. T.; Wiley, M. R.; Cordier-Lassalle, T.; Christie, A.; Schroth, G. P.; Gross, S. M.; Davies-Wayne, G. J.; Shinde, S. A.; Murugan, R.; Sieh, S. B.; Badio, M.; Fakoli, L.; Taweh, F.; de Wit, E.; van Doremalen, N.; Munster, V. J.; Pettitt, J.; Prieto, K.; Humrighouse, B. W.; Ströher, U.; DiClaro, J. W.; Hensley, L. E.; Schoepp, R. J.; Safronetz, D.; Fair, J.; Kuhn, J. H.; Blackley, D. J.; Laney, A. S.; Williams, D. E.; Lo, T.; Gasasira, A.; Nichol, S. T.; Formenty, P.; Kateh, F. N.; De Cock, K. M.; Bolay, F.; Sanchez-Lockhart, M.; Palacios, G. Molecular Evidence of Sexual Transmission of Ebola Virus. N. Engl. J. Med. 2015, 373, 2448– 2454, DOI: 10.1056/NEJMoa1509773Google Scholar4Molecular evidence of sexual transmission of Ebola virusMate, S. E.; Kugelman, J. R.; Nyenswah, T. G.; Ladner, J. T.; Wiley, M. R.; Cordier-Lassalle, T.; Christie, A.; Schroth, G. P.; Gross, S. M.; Davies-Wayne, G. J.; Shinde, S. A.; Murugan, R.; Sieh, S. B.; Badio, M.; Fakoli, L.; Taweh, F.; de Wit, E.; van Doremalen, N.; Munster, V. J.; Pettitt, J.; Prieto, K.; Humrighouse, B. W.; Stroher, U.; Di Claro, J. W.; Hensley, L. E.; Schoepp, R. J.; Safronetz, D.; Fair, J.; Kuhn, J. H.; Blackley, D. J.; Laney, A. S.; Williams, D. E.; Lo, T.; Gasasira, A.; Nichol, S. T.; Formenty, P.; Kateh, F. N.; De Cock, K. M.; Bolay, F.; Sanchez-Lockhart, M.; Palacios, G.New England Journal of Medicine (2015), 373 (25), 2448-2454CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiol. data, the genomic anal. provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD.
- 5Kuhn, J. H. Filoviruses: A Compendium of 40 Years of Epidemiological, Clinical, and Laboratory Studies; Calisher, C. H., Ed.; Springer: Wien, Austria, 2008.Google ScholarThere is no corresponding record for this reference.
- 6Rougeron, V.; Feldmann, H.; Grard, G.; Becker, S.; Leroy, E. M. Ebola and Marburg Haemorrhagic Fever. J. Clin. Virol. 2015, 64, 111– 119, DOI: 10.1016/j.jcv.2015.01.014Google Scholar6Ebola and Marburg haemorrhagic feverRougeron V; Feldmann H; Grard G; Becker S; Leroy E MJournal of clinical virology : the official publication of the Pan American Society for Clinical Virology (2015), 64 (), 111-9 ISSN:.Ebolaviruses and Marburgviruses (family Filoviridae) are among the most virulent pathogens for humans and great apes causing severe haemorrhagic fever and death within a matter of days. This group of viruses is characterized by a linear, non-segmented, single-stranded RNA genome of negative polarity. The overall burden of filovirus infections is minimal and negligible compared to the devastation caused by malnutrition and other infectious diseases prevalent in Africa such as malaria, dengue or tuberculosis. In this paper, we review the knowledge gained on the eco/epidemiology, the pathogenesis and the disease control measures for Marburg and Ebola viruses developed over the last 15 years. The overall progress is promising given the little attention that these pathogen have achieved in the past; however, more is to come over the next decade given the more recent interest in these pathogens as potential public and animal health concerns. Licensing of therapeutic and prophylactic options may be achievable over the next 5-10 years.
- 7Qiu, X.; Wong, G.; Audet, J.; Bello, A.; Fernando, L.; Alimonti, J. B.; Fausther-Bovendo, H.; Wei, H.; Aviles, J.; Hiatt, E.; Johnson, A.; Morton, J.; Swope, K.; Bohorov, O.; Bohorova, N.; Goodman, C.; Kim, D.; Pauly, M. H.; Velasco, J.; Pettitt, J.; Olinger, G. G.; Whaley, K.; Xu, B.; Strong, J. E.; Zeitlin, L.; Kobinger, G. P. Reversion af Advanced Ebola Virus Disease in Nonhuman Primates with ZMapp. Nature 2014, 514, 47– 53, DOI: 10.1038/nature13777Google Scholar7Reversion of advanced Ebola virus disease in nonhuman primates with ZMappQiu, Xiangguo; Wong, Gary; Audet, Jonathan; Bello, Alexander; Fernando, Lisa; Alimonti, Judie B.; Fausther-Bovendo, Hugues; Wei, Haiyan; Aviles, Jenna; Hiatt, Ernie; Johnson, Ashley; Morton, Josh; Swope, Kelsi; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Pauly, Michael H.; Velasco, Jesus; Pettitt, James; Olinger, Gene G.; Whaley, Kevin; Xu, Bianli; Strong, James E.; Zeitlin, Larry; Kobinger, Gary P.Nature (London, United Kingdom) (2014), 514 (7520), 47-53CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. A combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viremia and abnormalities in blood count and blood chem. were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal hemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clin. use.
- 8Thi, E. P.; Mire, C. E.; Lee, A. C. H.; Geisbert, J. B.; Zhou, J. Z.; Agans, K. N.; Snead, N. M.; Deer, D. J.; Barnard, T. R.; Fenton, K. A.; MacLachlan, I.; Geisbert, T. W. Lipid Nanoparticles siRNA Treatment of Ebola-Virus-Makona-Infected Nonhuman Primates. Nature 2015, 521, 362– 365, DOI: 10.1038/nature14442Google Scholar8Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primatesThi, Emily P.; Mire, Chad E.; Lee, Amy C. H.; Geisbert, Joan B.; Zhou, Joy Z.; Agans, Krystle N.; Snead, Nicholas M.; Deer, Daniel J.; Barnard, Trisha R.; Fenton, Karla A.; MacLachlan, Ian; Geisbert, Thomas W.Nature (London, United Kingdom) (2015), 521 (7552), 362-365CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viremic and clin. ill. Although all infected animals showed evidence of advanced disease including abnormal haematol., blood chem. and coagulopathy, siRNA-treated animals had milder clin. features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.
- 9Tekmira Pharmaceuticals Corp. Tekmira Provides Update on TKM-Ebola-Guinea. http://www.sec.gov/Archives/edgar/data/1447028/000117184315003522/newsrelease.htm, 2015 (accessed Jul. 22, 2016).Google ScholarThere is no corresponding record for this reference.
- 10Iversen, P. L.; Warren, T. K.; Wells, J. B.; Garza, N. L.; Mourich, D. V.; Welch, L. S.; Panchal, R. G.; Bavari, S. Discovery and Early Development of AVI-7537 and AVI-7288 For the Treatment of Ebola Virus and Marburg Virus Infections. Viruses 2012, 4, 2806– 2830, DOI: 10.3390/v4112806Google Scholar10Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infectionsIversen, Patrick L.; Warren, Travis K.; Wells, Jay B.; Garza, Nicole L.; Mourich, Dan V.; Welch, Lisa S.; Panchal, Rekha G.; Bavari, SinaViruses (2012), 4 (), 2806-2830CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus resp. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chem. modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus contg. a limited no. of pos. charged linkages in the PMO structure. The initial lead compds. were combinations of two agents targeting sep. genes. In the final anal., a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus and are now progressing into late stage clin. development as the optimal therapeutic candidates.
- 11Oestereich, L.; Lüdtke, A.; Wurr, S.; Rieger, T.; Muñoz-Fontela, C.; Günther, S. Successful Treatment of Advanced Ebola Virus Infection With T-705 (favipiravir) in a Small Animal Model. Antiviral Res. 2014, 105, 17– 21, DOI: 10.1016/j.antiviral.2014.02.014Google Scholar11Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal modelOestereich, Lisa; Ludtke, Anja; Wurr, Stephanie; Rieger, Toni; Munoz-Fontela, Cesar; Gunther, StephanAntiviral Research (2014), 105 (), 17-21CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Outbreaks of Ebola hemorrhagic fever in sub-Saharan Africa are assocd. with case fatality rates of up to 90%. Currently, neither a vaccine nor an effective antiviral treatment is available for use in humans. Here, we evaluated the efficacy of the pyrazinecarboxamide deriv. T-705 (favipiravir) against Zaire Ebola virus (EBOV) in vitro and in vivo. T-705 suppressed replication of Zaire EBOV in cell culture by 4 log units with an IC90 of 110 μM. Mice lacking the type I interferon receptor (IFNAR-/-) were used as in vivo model for Zaire EBOV-induced disease. Initiation of T-705 administration at day 6 post infection induced rapid virus clearance, reduced biochem. parameters of disease severity, and prevented a lethal outcome in 100% of the animals. The findings suggest that T-705 is a candidate for treatment of Ebola hemorrhagic fever.
- 12Smither, S. J.; Eastaugh, L. S.; Steward, J. A.; Nelson, M.; Lenk, R. P.; Lever, M. S. Post-exposure Efficacy of Oral T-705 (Favipiravir) Against Inhalational Ebola Virus Infection in a Mouse Model. Antiviral Res. 2014, 104, 153– 155, DOI: 10.1016/j.antiviral.2014.01.012Google Scholar12Post-exposure efficacy of Oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse modelSmither, Sophie J.; Eastaugh, Lin S.; Steward, Jackie A.; Nelson, Michelle; Lenk, Robert P.; Lever, Mark S.Antiviral Research (2014), 104 (), 153-155CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Filoviruses cause disease with high case fatality rates and are considered biol. threat agents. Licensed post-exposure therapies that can be administered by the oral route are desired for safe and rapid distribution and uptake in the event of exposure or outbreaks. Favipiravir or T-705 has broad antiviral activity and has already undergone phase II and is undergoing phase III clin. trials for influenza. Here we report the first use of T-705 against Ebola virus. T-705 gave 100% protection against aerosol Ebola virus E718 infection; protection was shown in immune-deficient mice after 14 days of twice-daily dosing. T-705 was also shown to inhibit Ebola virus infection in cell culture. T-705 is likely to be licensed for use against influenza in the near future and could also be used with a new indication for filovirus infection.
- 13Sissoko, D.; Folkesson, E.; Abdoul, M.; Beavogui, A. H.; Gunther, S.; Shepherd, S.; Danel, C.; Mentre, F.; Anglaret, X.; Malvy, D. Favipiravir in Patients With Ebola Virus Disease: Early Results of the JIKI Trial in Guinea. Conference on Retroviruses and Opportunistic Infections, Seattle, WA, USA, Feb. 23–26, 2015, Abstract 103-ALB; CROI Foundation/IAS-USA: San Francisco, CA, USA, 2015.Google ScholarThere is no corresponding record for this reference.
- 14McMullan, L. K.; Flint, M.; Dyall, J.; Albariño, C.; Olinger, G. G.; Foster, S.; Sethna, P.; Hensley, L. E.; Nichol, S. T.; Lanier, E. R.; Spiropoulou, C. F. The Lipid Moiety of Brincidofovir is Required For In Vitro Antiviral Activity Against Ebola Virus. Antiviral Res. 2016, 125, 71– 78, DOI: 10.1016/j.antiviral.2015.10.010Google Scholar14The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virusMcMullan, Laura K.; Flint, Mike; Dyall, Julie; Albarino, Cesar; Olinger, Gene G.; Foster, Scott; Sethna, Phiroze; Hensley, Lisa E.; Nichol, Stuart T.; Lanier, E. Randall; Spiropoulou, Christina F.Antiviral Research (2016), 125 (), 71-78CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was obsd. for several HDP-nucleotide conjugates.
- 15Warren, T. K.; Wells, J.; Panchal, R. G.; Stuthman, K. S.; Garza, N. L.; Van Tongeren, S. A.; Dong, L.; Retterer, C. J.; Eaton, B. P.; Pegoraro, G.; Honnold, S.; Bantia, S.; Kotian, P.; Chen, X.; Taubenheim, B. R.; Welch, L. S.; Minning, D. M.; Babu, Y. S.; Sheridan, W. P.; Bavari, S. Protection Against Filovirus Disease by a Novel Broad-Spectrum Nucleoside Analogue BCX4430. Nature 2014, 508, 402– 405, DOI: 10.1038/nature13027Google Scholar15Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430Warren, Travis K.; Wells, Jay; Panchal, Rekha G.; Stuthman, Kelly S.; Garza, Nicole L.; Van Tongeren, Sean A.; Dong, Lian; Retterer, Cary J.; Eaton, Brett P.; Pegoraro, Gianluca; Honnold, Shelley; Bantia, Shanta; Kotian, Pravin; Chen, Xilin; Taubenheim, Brian R.; Welch, Lisa S.; Minning, Dena M.; Babu, Yarlagadda S.; Sheridan, William P.; Bavari, SinaNature (London, United Kingdom) (2014), 508 (7496), 402-405CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analog, inhibits infection of distinct filoviruses in human cells. Biochem., reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure i.m. administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 h after infection. In addn., BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small mol. We provide addnl. pharmacol. characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.
- 16Henao-Restrepo, A. M.; Longini, I. M.; Egger, M.; Dean, N. E.; Edmunds, W. J.; Camacho, A.; Carroll, M. W.; Doumbia, M.; Draguez, B.; Duraffour, S.; Enwere, G.; Grais, R.; Gunther, S.; Hossmann, S.; Kondé, M. K.; Kone, S.; Kuisma, E.; Levine, M. M.; Mandal, S.; Norheim, G.; Riveros, X.; Soumah, A.; Trelle, S.; Vicari, A. S.; Watson, C. H.; Kéïta, S.; Kieny, M. P.; Røttingen, J.-A. Efficacy and Effectiveness of an rVSV-Vectored Vaccine Expressing Ebola, Surface Glycoprotein: Interim Results From the Guinea Ring Vaccination Cluster-Randomised Trial. Lancet 2015, 386, 857– 866, DOI: 10.1016/S0140-6736(15)61117-5Google Scholar16Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trialHenao-Restrepo, Ana Maria; Longini, Ira M.; Egger, Matthias; Dean, Natalie E.; Edmunds, W. John; Camacho, Anton; Carroll, Miles W.; Doumbia, Moussa; Draguez, Bertrand; Duraffour, Sophie; Enwere, Godwin; Grais, Rebecca; Gunther, Stephan; Hossmann, Stefanie; Konde, Mandy Kader; Kone, Souleymane; Kuisma, Eeva; Levine, Myron M.; Mandal, Sema; Norheim, Gunnstein; Riveros, Ximena; Soumah, Aboubacar; Trelle, Sven; Vicari, Andrea S.; Watson, Conall H.; Keita, Sakoba; Kieny, Marie Paule; Roettingen, John-ArneLancet (2015), 386 (9996), 857-866CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)A recombinant, replication-competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Zaire Ebolavirus (rVSV-ZEBOV) is a promising Ebola vaccine candidate. We report the results of an interim anal. of a trial of rVSV-ZEBOV in Guinea, west Africa. For this open-label, cluster-randomised ring vaccination trial, suspected cases of Ebola virus disease (Guinea, west Africa) were independently ascertained by Ebola response teams as part of a national surveillance system. After lab. confirmation of a new case, clusters of all contacts and contacts of contacts were defined and randomly allocated 1:1 to immediate vaccination or delayed (21 days later) vaccination with rVSV-ZEBOV (one dose of 2 × 107 plaque-forming units, administered i.m. in the deltoid muscle). Adults (age ≥18 years) who were not pregnant or breastfeeding were eligible for vaccination. Block randomisation was used, with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 vs >20 individuals). The study is open label and masking of participants and field teams to the time of vaccination is not possible, but Ebola response teams and lab. workers were unaware of allocation to immediate or delayed vaccination. Taking into account the incubation period of the virus of about 10 days, the prespecified primary outcome was lab.-confirmed Ebola virus disease with onset of symptoms at least 10 days after randomisation. The primary anal. was per protocol and compared the incidence of Ebola virus disease in eligible and vaccinated individuals in immediate vaccination clusters with the incidence in eligible individuals in delayed vaccination clusters. This trial is registered with the Pan African Clin. Trials Registry, no. PACTR201503001057193. Between Apr. 1, 2015, and July 20, 2015, 90 clusters, with a total population of 7651 people were included in the planned interim anal. 48 of these clusters (4123 people) were randomly assigned to immediate vaccination with rVSV-ZEBOV, and 42 clusters (3528 people) were randomly assigned to delayed vaccination with rVSV-ZEBOV. In the immediate vaccination group, there were no cases of Ebola virus disease with symptom onset at least 10 days after randomisation, whereas in the delayed vaccination group there were 16 cases of Ebola virus disease from seven clusters, showing a vaccine efficacy of 100% (95% CI 74·7-100·0; p=0·0036). No new cases of Ebola virus disease were diagnosed in vaccinees from the immediate or delayed groups from 6 days post-vaccination. At the cluster level, with the inclusion of all eligible adults, vaccine effectiveness was 75·1% (95% CI -7·1 to 94·2; p=0·1791), and 76·3% (95% CI -15·5 to 95·1; p=0·3351) with the inclusion of everyone (eligible or not eligible for vaccination). 43 serious adverse events were reported; one serious adverse event was judged to be causally related to vaccination (a febrile episode in a vaccinated participant, which resolved without sequelae). Assessment of serious adverse events is ongoing. The results of this interim anal. indicate that rVSV-ZEBOV might be highly efficacious and safe in preventing Ebola virus disease, and is most likely effective at the population level when delivered during an Ebola virus disease outbreak via a ring vaccination strategy.
- 17Warren, T. K.; Jordan, R.; Lo, M. K.; Ray, A. S.; Mackman, R. L.; Soloveva, V.; Siegel, D.; Perron, M.; Bannister, R.; Hui, H. C.; Larson, N.; Strickley, R.; Wells, J.; Stuthman, K. S.; Van Tongeren, S. A.; Garza, N. L.; Donnelly, G.; Shurtleff, A. C.; Retterer, C. J.; Gharaibeh, D.; Zamani, R.; Kenny, T.; Eaton, B. P.; Grimes, E.; Welch, L. S.; Gomba, L.; Wilhelmsen, C. L.; Nichols, D. K.; Nuss, J. E.; Nagle, E. R.; Kugelman, J. R.; Palacios, G.; Doerffler, E.; Neville, S.; Carra, E.; Clarke, M. O.; Zhang, L.; Lew, W.; Ross, B.; Wang, Q.; Chun, K.; Wolfe, L.; Babusis, D.; Park, Y.; Stray, K. M.; Trancheva, I.; Feng, J. Y.; Barauskas, O.; Xu, Y.; Wong, P.; Braun, M. R.; Flint, M.; McMullan, L. K.; Chen, S. S.; Fearns, R.; Swaminathan, S.; Mayers, D. L.; Spiropoulou, C. F.; Lee, W. A.; Nichol, S. T.; Cihlar, T.; Bavari, S. Therapeutic Efficacy of The Small Molecule GS-5734 Against Ebola Virus in Rhesus Monkeys. Nature 2016, 531, 381– 385, DOI: 10.1038/nature17180Google Scholar17Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeysWarren, Travis K.; Jordan, Robert; Lo, Michael K.; Ray, Adrian S.; Mackman, Richard L.; Soloveva, Veronica; Siegel, Dustin; Perron, Michel; Bannister, Roy; Hui, Hon C.; Larson, Nate; Strickley, Robert; Wells, Jay; Stuthman, Kelly S.; Van Tongeren, Sean A.; Garza, Nicole L.; Donnelly, Ginger; Shurtleff, Amy C.; Retterer, Cary J.; Gharaibeh, Dima; Zamani, Rouzbeh; Kenny, Tara; Eaton, Brett P.; Grimes, Elizabeth; Welch, Lisa S.; Gomba, Laura; Wilhelmsen, Catherine L.; Nichols, Donald K.; Nuss, Jonathan E.; Nagle, Elyse R.; Kugelman, Jeffrey R.; Palacios, Gustavo; Doerffler, Edward; Neville, Sean; Carra, Ernest; Clarke, Michael O.; Zhang, Lijun; Lew, Willard; Ross, Bruce; Wang, Queenie; Chun, Kwon; Wolfe, Lydia; Babusis, Darius; Park, Yeojin; Stray, Kirsten M.; Trancheva, Iva; Feng, Joy Y.; Barauskas, Ona; Xu, Yili; Wong, Pamela; Braun, Molly R.; Flint, Mike; McMullan, Laura K.; Chen, Shan-Shan; Fearns, Rachel; Swaminathan, Swami; Mayers, Douglas L.; Spiropoulou, Christina F.; Lee, William A.; Nichol, Stuart T.; Cihlar, Tomas; Bavari, SinaNature (London, United Kingdom) (2016), 531 (7594), 381-385CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The most recent Ebola virus outbreak in West Africa, which was unprecedented in the no. of cases and fatalities, geog. distribution, and no. of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clin. efficacy. Here we report the discovery of a novel small mol. GS-5734, a monophosphoramidate prodrug of an adenosine analog, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacol. active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. I.v. administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily i.v. administration of 10 mg kg-1 GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clin. disease signs and pathophysiol. markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-mol. antiviral compd. against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufg., and clin. studies investigating the drug safety and pharmacokinetics are ongoing.
- 18Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy Phosphoramidate Triesters: A Technology For Delivering Monophosphorylated Nucleosides and Sugars Into Cells. ChemMedChem 2009, 4, 1779– 1791, DOI: 10.1002/cmdc.200900289Google Scholar18Aryloxy Phosphoramidate Triesters: a Technology for Delivering Monophosphorylated Nucleosides and Sugars into CellsMehellou, Youcef; Balzarini, Jan; McGuigan, ChristopherChemMedChem (2009), 4 (11), 1779-1791CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Prodrug technologies aimed at delivering nucleoside monophosphates into cells (protides) have proved to be effective in improving the therapeutic potential of antiviral and anticancer nucleosides. In these cases, the nucleoside monophosphates are delivered into the cell, where they may then be further converted (phosphorylated) to their active species. Herein, we describe one of these technologies developed in our labs., known as the phosphoramidate protide method. In this approach, the charges of the phosphate group are fully masked to provide efficient passive cell-membrane penetration. Upon entering the cell, the masking groups are enzymically cleaved to release the phosphorylated biomol. The application of this technol. to various therapeutic nucleosides has resulted in improved antiviral and anticancer activities, and in some cases it has transformed inactive nucleosides to active ones. Addnl., the phosphoramidate technol. has also been applied to numerous antiviral nucleoside phosphonates, and has resulted in at least three phosphoramidate-based nucleotides progressing to clin. investigations. Furthermore, the phosphoramidate technol. has been recently applied to sugars (mainly glucosamine) in order to improve their therapeutic potential. The development of the phosphoramidate technol., mechanism of action and the application of the technol. to various monophosphorylated nucleosides and sugars will be reviewed.
- 19Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.; Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang, H.-R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a β-D-2′-Deoxy-2′-α-Fluoro-2′-β-C-Methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C Virus. J. Med. Chem. 2010, 53, 7202– 7218, DOI: 10.1021/jm100863xGoogle Scholar19Discovery of a β-D-2'-Deoxy-2'-α-fluoro-2'-β-C-methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C VirusSofia, Michael J.; Bao, Donghui; Chang, Wonsuk; Du, Jinfa; Nagarathnam, Dhanapalan; Rachakonda, Suguna; Reddy, P. Ganapati; Ross, Bruce S.; Wang, Peiyuan; Zhang, Hai-Ren; Bansal, Shalini; Espiritu, Christine; Keilman, Meg; Lam, Angela M.; Steuer, Holly M. Micolochick; Niu, Congrong; Otto, Michael J.; Furman, Phillip A.Journal of Medicinal Chemistry (2010), 53 (19), 7202-7218CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. β-D-2'-Deoxy-2'-α-fluoro-2'-β-C-Me nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5'-phosphate deriv. of the β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methyluridine nucleoside were prepd. and showed significant potency in the HCV subgenomic replicon assay (<1 μM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixt. 14 was crystd., and an X-ray structure was detd. establishing the phosphoramidate stereochem. as Sp, thus correlating for the first time the stereochem. of a phosphoramidate prodrug with biol. activity. 51 (PSI-7977) was selected as a clin. development candidate.
- 20Lee, A. W.; He, G.-X.; Eisenberg, E.; Cihlar, T.; Swaminathan, S.; Mulato, A.; Cundy, K. C. Selective Intracellular Activation of a Novel Prodrug of the Human Immunodeficiency Virus Reverse Transcriptase Inhibitor Tenofovir Leads to Preferential Distribution and Accumulation in Lymphatic Tissue. Antimicrob. Agents Chemother. 2005, 49, 1898– 1906, DOI: 10.1128/AAC.49.5.1898-1906.2005Google Scholar20Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissueLee, William A.; He, Gong-Xin; Eisenberg, Eugene; Cihlar, Tomas; Swaminathan, Swami; Mulato, Andrew; Cundy, Kenneth C.Antimicrobial Agents and Chemotherapy (2005), 49 (5), 1898-1906CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)An isopropylalaninyl monoamidate Ph monoester prodrug of tenofovir (GS 7340) was prepd., and its in vitro antiviral activity, metab., and pharmacokinetics in dogs were detd. The 50% effective concn. (EC50) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 μM compared to an EC50 of 5 μM for the parent drug, tenofovir. The (L)-alaninyl analog (GS 7340) was >1000-fold more active than the (D)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at 37° and a half-life of 28.3 min in an MT-2 cell ext. at 37°. The antiviral activity (>10× the EC50) and the metabolic stability in MT-2 cell exts. (>35×) and plasma (>2.5×) were also sensitive to the stereochem. at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs, the plasma bioavailability of tenofovir compared to an i.v. dose of tenofovir was 17%. The total intracellular concn. of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was 63 μg-eq/mL compared to 0.2 μg-eq/mL in plasma. A radiolabeled distribution study with dogs resulted in an increased distribution of tenofovir to tissues of lymphatic origin compared to the com. available prodrug tenofovir DF (Viread).
- 21Murakami, E.; Niu, C.; Bao, H.; Steuer, H. M. M.; Whitaker, T.; Nachman, T.; Sofia, M. A.; Wang, P.; Otto, M. J.; Furman, P. A. The Mechanism of Action of β-D-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine Involves a Second Metabolic Pathway Leading to β-D-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine 5′-Triphosphate, A Potent Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase. Antimicrob. Agents Chemother. 2008, 52, 458– 464, DOI: 10.1128/AAC.01184-07Google Scholar21The mechanism of action of β-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine involves a second metabolic pathway leading to β-D-2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-triphosphate, a potent inhibitor of the hepatitis C virus RNA-dependent RNA polymeraseMurakami, Eisuke; Niu, Congrong; Bao, Haiying; Micolochick Steuer, Holly M.; Whitaker, Tony; Nachman, Tammy; Sofia, Michael A.; Wang, Peiyuan; Otto, Michael J.; Furman, Phillip A.Antimicrobial Agents and Chemotherapy (2008), 52 (2), 458-464CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)β-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metab. of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, β-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, resp. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.
- 22Cho, A.; Saunders, O. L.; Butler, T.; Zhang, L.; Xu, J.; Vela, J. E.; Feng, J. Y.; Ray, A. S.; Kim, C. U. Synthesis and Antiviral Activity of a Series of 1′-Substituted 4-Aza-7,9-Dideazaadenosine C-Nucleosides. Bioorg. Med. Chem. Lett. 2012, 22, 2705– 2707, DOI: 10.1016/j.bmcl.2012.02.105Google Scholar22Synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine C-nucleosidesCho, Aesop; Saunders, Oliver L.; Butler, Thomas; Zhang, Lijun; Xu, Jie; Vela, Jennifer E.; Feng, Joy Y.; Ray, Adrian S.; Kim, Choung U.Bioorganic & Medicinal Chemistry Letters (2012), 22 (8), 2705-2707CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)A series of 1'-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside, e.g. I, were prepd. and evaluated for the potential as antiviral agents. These compds. showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R = CN was attributed to inhibition of HCV NS5B polymerase and intracellular concn. of the corresponding nucleoside triphosphate.
- 23Mackman, R. L.; Parrish, J. P.; Ray, A. S.; Theodore, D. A. Methods and Compounds for Treating Paramyxoviridae Virus Infections. U.S. Patent 2011045102 July, 22, 2011.Google ScholarThere is no corresponding record for this reference.
- 24Patil, S. A.; Otter, P. B.; Klein, R. S. 4-Aza-7,9-Dideazaadenosine, A New Cytotoxic Synthetic C-Nucleoside Analogue of Adenosine. Tetrahedron Lett. 1994, 35, 5339– 5342, DOI: 10.1016/S0040-4039(00)73494-0Google Scholar244-Aza-7,9-dideazaadenosine, a new cytotoxic synthetic C-nucleoside analog of adenosinePatil, Shirish A.; Otter, Brian A.; Klein, Robert S.Tetrahedron Letters (1994), 35 (30), 5339-42CODEN: TELEAY; ISSN:0040-4039.The first synthesis of I, the pyrrolo[2,1-f]triazine C-nucleoside congener of adenosine is described. The key intermediate ribofuranosyl pyrrole is obtained by the direct C-ribosylation of pyrrolemagnesium bromide followed by an acid-catalyzed dehydration, Vilsmeier formylation, and N-amination. In vitro growth inhibitory activities of I against leukemic cell lines (0.8-15 nM) are comparable to those of 9-deazaadenosine.
- 25Lou, Z.; Chen, G.; Xie, Y. Cyanoribofuranoside Compound and a Preparation Method Thereof. Chinese Patent CN 1137132 C Feb., 4, 2004.Google ScholarThere is no corresponding record for this reference.
- 26Yoshimura, Y.; Kano, F.; Miyazaki, S.; Ashida, N.; Sakata, S.; Haraguchi, K.; Itoh, Y.; Tanaka, H.; Miyasaka, T. Synthesis and Biological Evaluation of 1′-C-Cyano-Pyrimidine Nucleosides. Nucleosides, Nucleotides Nucleic Acids 1996, 15, 305– 324, DOI: 10.1080/07328319608002386Google ScholarThere is no corresponding record for this reference.
- 27Kirschberg, T. A.; Mish, M.; Squires, N. H.; Zonte, S.; Aktoudianakis, E.; Metobo, S.; Butler, T.; Ju, X.; Cho, A.; Ray, A. S.; Kim, C. U. Synthesis of 1′-C-Cyano Pyrimidine Nucleosides and Characterization as HCV Polymerase Inhibitors. Nucleosides, Nucleotides Nucleic Acids 2015, 34, 763– 785, DOI: 10.1080/15257770.2015.1075550Google Scholar27Synthesis of 1'-C-Cyano Pyrimidine Nucleosides and Characterization as HCV Polymerase InhibitorsKirschberg, Thorsten A.; Mish, Michael; Squires, Neil H.; Zonte, Sebastian; Aktoudianakis, Evangelos; Metobo, Sammy; Butler, Thomas; Ju, Xie; Cho, Aesop; Ray, Adrian S.; Kim, Choung U.Nucleosides, Nucleotides & Nucleic Acids (2015), 34 (11), 763-785CODEN: NNNAFY; ISSN:1525-7770. (Taylor & Francis Ltd.)Ribose modified 1'-C-cyano pyrimidine nucleosides were synthesized. A silver triflate mediated Vorbruggen reaction was used to generate the nucleoside scaffold and follow-up chem. provided specific ribose modified analogs. Nucleosides and phosphoramidate prodrugs were tested for their anti-HCV activity.
- 28Cho, A.; Zhang, L.; Xu, J.; Lee, R.; Butler, T.; Metobo, S.; Aktoudianakis, V.; Lew, W.; Ye, H.; Clarke, M.; Doerffler, E.; Byun, D.; Wang, T.; Babusis, D.; Carey, A. C.; German, P.; Sauer, D.; Zhong, W.; Rossi, S.; Fenaux, M.; McHutchison, J. G.; Perry, J.; Feng, J.; Ray, A. S.; Kim, C. U. Discovery of the First C-nucleoside HCV Polymerase Inhibitor (GS-6620) with Demonstrated Antiviral Response in HCV Infected Patients. J. Med. Chem. 2014, 57, 1812– 1825, DOI: 10.1021/jm400201aGoogle Scholar28Discovery of the First C-Nucleoside HCV Polymerase Inhibitor (GS-6620) with Demonstrated Antiviral Response in HCV Infected PatientsCho, Aesop; Zhang, Lijun; Xu, Jie; Lee, Rick; Butler, Thomas; Metobo, Sammy; Aktoudianakis, Vangelis; Lew, Willard; Ye, Hong; Clarke, Michael; Doerffler, Edward; Byun, Daniel; Wang, Ting; Babusis, Darius; Carey, Anne C.; German, Polina; Sauer, Dorothea; Zhong, Weidong; Rossi, Stephen; Fenaux, Martijn; McHutchison, John G.; Perry, Jason; Feng, Joy; Ray, Adrian S.; Kim, Choung U.Journal of Medicinal Chemistry (2014), 57 (5), 1812-1825CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clin. development thus far have all been N-nucleoside derivs. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-Me 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compds. showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clin. development candidate GS-6620 (I). In a phase I clin. study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.
- 29(a) Smith, J. T.; Elkin, J. T.; Reichert, W. M. Directed Cell Migration on Fibronectin Gradients: Effect of Gradient Slope. Exp. Cell Res. 2006, 312, 2424– 2432, DOI: 10.1016/j.yexcr.2006.04.005Google Scholar29aDirected cell migration on fibronectin gradients: Effect of gradient slopeSmith, Jason T.; Elkin, James T.; Reichert, W. MontyExperimental Cell Research (2006), 312 (13), 2424-2432CODEN: ECREAL; ISSN:0014-4827. (Elsevier)The migration of human microvascular endothelial cells (hMEC) was measured on a range of fibronectin gradient slopes. hMEC drift speed increased with increasing gradient slope with no concurrent change in cellular persistence time or random cell speed. The frequency of discrete cellular motion in the gradient direction increased with gradient slope. Morphol. polarization of cells on the gradients is also characterized and correlated with cellular drift speed. These expts. present the first demonstration of cellular response to changing haptotactic gradient slope using an in vitro system for the quant. study of cell migration.(b) Zhao, L.; Kroenke, C. D.; Song, J.; Piwnica-Worms, D.; Ackerman, J. J. H.; Neil, J. J. Intracellular Water Specific MR of Microbead-Adherent Cells: The HeLa Cell Intracellular Water Exchange Lifetime. NMR Biomed. 2008, 21, 159– 164, DOI: 10.1002/nbm.1173Google Scholar29bIntracellular water-specific MR of microbead-adherent cells: the HeLa cell intracellular water exchange lifetimeZhao, L.; Kroenke, C. D.; Song, J.; Piwnica-Worms, D.; Ackerman, J. J. H.; Neil, J. J.NMR in Biomedicine (2008), 21 (2), 159-164CODEN: NMRBEF; ISSN:0952-3480. (John Wiley & Sons Ltd.)Quant. characterization of the intracellular water 1H MR signal from cultured cells will provide crit. biophys. insight into the MR signal from tissues in vivo. Microbeads provide a robust immobilization substrate for the m any mammalian cell lines that adhere to surfaces and also provide sufficient cell d. for observation of the intracellular water MR signal. However, selective observation of the intracellular water MR signal from perfused, microbead-adherent mammalian cells requires highly effective suppression of the extracellular water MR signal. We describe how high-velocity perfusion of microbead-adherent cells results in short apparent 1H MR longitudinal and transverse relaxation times for the extracellular water in a thin slice selected orthogonal to the direction of flow. When combined with a spin-echo pulse sequence, this phenomenon provides highly effective suppression of the extracellular water MR signal. This new method is exploited here to quantify the kinetics of water exchange from the intracellular to extracellular spaces of HeLa cells. The time const. describing water exchange from intracellular to extracellular spaces, also known as the exchange lifetime for intracellular water, is 119 ± 14 ms.
- 30Clarke, M. O.; Mackman, R.; Byun, D.; Hui, H.; Barauskas, O.; Birkus, G.; Chun, B.-K.; Doerffler, E.; Feng, J.; Karki, K.; Lee, G.; Perron, M.; Siegel, D.; Swaminathan, S.; Lee, W. Discovery of β-D-2′-α-Fluoro-4′-α-Cyano-5-Aza-7,9-Dideaza Adenosine as a Potent Nucleoside Inhibitor of Respiratory Syncytial Virus With Excellent Selectivity Over Mitochondrial RNA and DNA Polymerases. Bioorg. Med. Chem. Lett. 2015, 25, 2484– 2487, DOI: 10.1016/j.bmcl.2015.04.073Google Scholar30Discovery of β-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerasesClarke, Michael O.; Mackman, Richard; Byun, Daniel; Hui, Hon; Barauskas, Ona; Birkus, Gabriel; Chun, Byoung-Kwon; Doerffler, Edward; Feng, Joy; Karki, Kapil; Lee, Gary; Perron, Michel; Siegel, Dustin; Swaminathan, Swami; Lee, WilliamBioorganic & Medicinal Chemistry Letters (2015), 25 (12), 2484-2487CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Novel 4'-substituted β-D-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compds. demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of I as a promising nucleoside lead for RSV.
- 31Feng, J.; Xu, Y.; Barauskas, O.; Perry, J. K.; Ahmadyar, S.; Stepan, G.; Yu, H.; Babusis, D.; Park, Y.; McCutcheon, K.; Perron, M.; Schultz, B. E.; Sakowicz, R.; Ray, A. S. Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus. Antimicrob. Agents Chemother. 2016, 60, 806– 817, DOI: 10.1128/AAC.01922-15Google Scholar31Role of mitochondrial RNA polymerase in the toxicity of nucleotide inhibitors of hepatitis C virusFeng, Joy Y.; Xu, Yili; Barauskas, Ona; Perry, Jason K.; Ahmadyar, Shekeba; Stepan, George; Yu, Helen; Babusis, Darius; Park, Yeojin; McCutcheon, Krista; Perron, Michel; Schultz, Brian E.; Sakowicz, Roman; Ray, Adrian S.Antimicrobial Agents and Chemotherapy (2016), 60 (2), 806-817CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)Toxicity has emerged during the clin. development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clin. relevant HCV NI were characterized in biochem. and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4'-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2'-C-Me guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concns., followed by general cytotoxicity. In contrast, NI contg. multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2'-deoxynucleotide analogs, there is an assocn. between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clin. testing.
- 32(a) Müller, R.; Poch, O.; Delarue, M.; Bishop, D. H. L.; Bouloy, M. Rift Valley Fever Virus L Segment: Correction of the Sequence and Possible Functional Role of Newly Identified Regions Conserved in RNA-Dependent Polymerases. J. Gen. Virol. 1994, 75, 1345– 1352, DOI: 10.1099/0022-1317-75-6-1345Google Scholar32aRift valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerasesMueller, R.; Poch, O.; Delarue, M.; Bishop, D. H. L.; Bouloy, M.Journal of General Virology (1994), 75 (6), 1345-52CODEN: JGVIAY; ISSN:0022-1317.The sequence of Rift Valley fever virus L segment that the authors published in a previous paper was erroneous in the 3'-terminal region of the antigenomic RNA mol. Here, the authors have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237.7 K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 neg.-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called polymerase module, the authors identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented neg.-stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA-dependent polymerases in the polymerase module, the authors have detd. the position of the conserved residues in these polymerases and discuss their possible functions in light of the variable structural information.(b) Huang, H.; Chopra, R.; Verdine, G. L.; Harrison, S. C. Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistance. Science 1998, 282, 1669– 1675, DOI: 10.1126/science.282.5394.1669Google Scholar32bStructure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistanceHuang, Huifang; Chopra, Rajiv; Verdine, Gregory L.; Harrison, Stephen C.Science (Washington, D. C.) (1998), 282 (5394), 1669-1675CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A combinatorial disulfide crosslinking strategy was used to prep. a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase with a DNA template:primer and a deoxynucleoside triphosphate (dNTP), and the crystal structure of the complex was detd. at a resoln. of 3.2 angstroms. The presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the mol. basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chem. structure of the drug.(c) Appleby, T. C.; Perry, J. K.; Murakami, E.; Barauskas, O.; Feng, J.; Cho, A.; Fox, D.; Wetmore, D. R.; McGrath, M. E.; Ray, A. S.; Sofia, M. J.; Swaminathan, S.; Edwards, T. E. Structural Basis For RNA Replication by the Hepatitis C Virus Polymerase. Science 2015, 347, 771– 775, DOI: 10.1126/science.1259210Google Scholar32cStructural basis for RNA replication by the hepatitis C virus polymeraseAppleby, Todd C.; Perry, Jason K.; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David, III; Wetmore, Diana R.; McGrath, Mary E.; Ray, Adrian S.; Sofia, Michael J.; Swaminathan, S.; Edwards, Thomas E.Science (Washington, DC, United States) (2015), 347 (6223), 771-775CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Nucleotide analog inhibitors have shown clin. success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by detg. crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our anal. revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clin. active metabolite formed by sofosbuvir, to elucidate key mol. interactions in the active site.
- 33
For active site models of other filovirus polymerases see the Supporting Information.
There is no corresponding record for this reference. - 34Feng, J. Y.; Cheng, G.; Perry, J.; Barauskas, O.; Xu, Y.; Fenaux, M.; Eng, S.; Tirunagari, N.; Peng, B.; Yu, M.; Tian, Y.; Lee, Y.-J.; Stepan, G.; Lagpacan, L. L.; Jin, D.; Hung, M.; Ku, K. S.; Han, B.; Kitrinos, K.; Perron, M.; Birkus, G.; Wong, K. A.; Zhong, W.; Kim, C. U.; Carey, A.; Cho, A.; Ray, A. S. Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C-Nucleoside Monophosphate Prodrug. Antimicrob. Agents Chemother. 2014, 58, 1930– 1942, DOI: 10.1128/AAC.02351-13Google Scholar34Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrugFeng, Joy Y.; Cheng, Guofeng; Perry, Jason; Barauskas, Ona; Xu, Yili; Fenaux, Martijn; Eng, Stacey; Tirunagari, Neeraj; Peng, Betty; Yu, Mei; Tian, Yang; Lee, Yu-Jen; Stepan, George; Lagpacan, Leanna L.; Jin, Debi; Hung, Magdeleine; Ku, Karin S.; Han, Bin; Kitrinos, Kathryn; Perron, Michel; Birkus, Gabriel; Wong, Kelly A.; Zhong, Weidong; Kim, Choung U.; Carey, Anne; Cho, Aesop; Ray, Adrian S.Antimicrobial Agents and Chemotherapy (2014), 58 (4), 1930-1942, 14 pp.CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacol. of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concn. [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, resp. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clin. trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.
- 35Butler, T.; Cho, A.; Kim, C. U.; Saunders, O. L.; Zhang, L. 1′-Substituted Carba-Nucleoside Analogs for Antiviral Treatment. U.S. Patent 2009041447 Apr., 22, 2009.Google ScholarThere is no corresponding record for this reference.
- 36Butler, T.; Cho, A.; Graetz, B. R.; Kim, C. U.; Metobo, S. E.; Saunders, O. L.; Waltman, A. W.; Xu, J.; Zhang, L. Processes and Intermediates for the Preparation of 1′-Substituted Carba-Nucleoside Analogs. U.S. Patent20100459508 Sep., 20, 2010.Google ScholarThere is no corresponding record for this reference.
- 37Metobo, S. E.; Xu, J.; Saunders, O. L.; Butler, T.; Aktoudianakis, E.; Cho, A.; Kim, C. U. Practical Synthesis of 1′-Substituted Tubercidin C-Nucleoside Analogs. Tetrahedron Lett. 2012, 53, 484– 486, DOI: 10.1016/j.tetlet.2011.11.055Google Scholar37Practical synthesis of 1'-substituted Tubercidin C-nucleoside analogsMetobo, Sammy E.; Xu, Jie; Saunders, Oliver L.; Butler, Thomas; Aktoudianakis, Evangelos; Cho, Aesop; Kim, Choung U.Tetrahedron Letters (2012), 53 (5), 484-486CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)Several 1'-substituted analogs of Tubercidin C-nucleosides were prepd. using a highly convergent synthesis. Good to high diastereoselectivity was achieved using a variety of nucleophiles targeting the 1'-position. The source for this stereoselectivity is herein proposed. It is thought to be attributed to a temp.-dependent chelation of the incoming nucleophile to either the 2'- or 3'-benzyloxy ether of the ribose core.
- 38Axt, S. D.; Badalov, P. R.; Brak, K.; Campagna, S.; Chtchemelinine, A.; Chun, B. K.; Clarke, M. O. H.; Doerffler, E.; Frick, M. M.; Gao, D.; Heumann, L. V.; Hoang, B.; Hui, H. C.; Jordan, R.; Lew, W.; Mackman, R. L.; Milburn, R. R.; Neville, S. T.; Parrish, J. P.; Ray, A. S.; Ross, B.; Rueden, E.; Scott, R. W.; Siegel, D.; Stevens, A. C.; Tadeus, C.; Vieira, T.; Waltman, A. W.; Wang, X.; Whitcomb, M. C.; Wolfe, L.; Yu, C.-Y. Methods For Treating Filoviridae Virus Infections. U.S. Patent 2015017934, Oct. 29, 2015.Google ScholarThere is no corresponding record for this reference.
- 39Krasovskiy, A.; Knochel, P. A LiCl-Mediated Br/Mg Exchange Reaction For the Preparation of Functionalized Aryl- and Heteroarylmagnesium Compounds From Organic Bromides. Angew. Chem., Int. Ed. 2004, 43, 3333– 3336, DOI: 10.1002/anie.200454084Google Scholar39A LiCl-mediated Br/Mg exchange reaction for the preparation of functionalized aryl- and heteroarylmagnesium compounds from organic bromidesKrasovskiy, Arkady; Knochel, PaulAngewandte Chemie, International Edition (2004), 43 (25), 3333-3336CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A wide range of aryl and heteroaryl bromides, which are usually sluggish in exchange reactions, are readily converted into the corresponding Grignard reagents by means of a Br/Mg exchange reaction triggered by iPrMgCl·LiCl. These Grignard intermediates react with electrophiles in good yields. For example, 4-MeOC6H4MgCl·LiCl was prepd. from iPrMgCl·LiCl and 4-MeOC6H4Br and reacted with PhCHO giving 4-MeOC6H4CH(OH)Ph (70% yield).
- 40For a separate account describing the crystallization induced resolution of p-nitrophenolate 2-ethylbutyl-L-alaninate phosphoramidate see: Klasson, B.; Eneroth, A.; Nilson, M.; Pinho, P.; Samuelsson, B.; Sund, C. HCV Polymerase Inhibitors. European Patent IB2012056994 Dec. 5, 2012. The conditions in this patent were identified independently/concurrently with this report.Google ScholarThere is no corresponding record for this reference.
- 41Bahar, F. G.; Ohura, K.; Ogihara, T.; Imai, T. Species Difference of Esterase Expression and Hydrolase Activity in Plasma. J. Pharm. Sci. 2012, 101, 3979– 3988, DOI: 10.1002/jps.23258Google Scholar41Species difference of esterase expression and hydrolase activity in plasmaBahar, Fatma Goksin; Ohura, Kayoko; Ogihara, Takuo; Imai, TerukoJournal of Pharmaceutical Sciences (2012), 101 (10), 3979-3988CODEN: JPMSAE; ISSN:0022-3549. (John Wiley & Sons, Inc.)Differences in esterase expression among human, rhesus monkey, cynomolgus monkey, dog, minipig, rabbit, rat, and mouse plasma were identified using native polyacrylamide gel electrophoresis. Paraoxonase (PON) and butyrylcholinesterase (BChE) were ubiquitous in all species, but were highly expressed in primates and dogs, whereas carboxylesterase (CES) was only abundant in rabbits, mice, and rats. Several unknown esterases were obsd. in minipig and mouse plasma. These differences in plasma esterases and their expression levels result in species differences with respect to hydrolase activity. These differences were characterized using several different substrates. In contrast to the high hydrolase activity found for p-nitrophenylacetate (PNPA), a substrate of several hydrolase enzymes, irinotecan, a carbamate compd., was resistant to all plasma esterases. Oseltamivir, temocapril, and propranolol (PL) derivs. were rapidly hydrolyzed in mouse and rat plasma by their highly active CES enzyme, but rabbit plasma CES hydrolyzed only the PL derivs. Interestingly, PL derivs. were highly hydrolyzed by monkey plasma BChE, whereas BChE from human, dog, and minipig plasma showed negligible activity. In conclusion, the esterase expression and hydrolyzing pattern of dog plasma were found to be closest to that of human plasma. These differences should be considered when selecting model animals for preclin. studies.
- 42Jacobs, M.; Rodger, A.; Bell, D. D.; Bhagani, S.; Cropley, I.; Filipe, A.; Gifford, R. J.; Hopkins, S.; Hughes, J.; Jabeen, F.; Johannessen, I.; Karageorgopoulos, D.; Lackenby, A.; Lester, R.; Liu, R. S. N.; MacConnachie, A.; Mahungu, T.; Martin, D.; Marshall, N.; Mepham, S.; Orton, R.; Palmarini, M.; Patel, M.; Perry, C.; Peters, S. E.; Porter, D.; Ritchie, D.; Ritchie, N. D.; Seaton, R. A.; Sreenu, V. B.; Templeton, K.; Warren, S.; Wilkie, G. S.; Zambon, M.; Gopal, R.; Thomson, E. C. Late Ebola Virus Relapse Causing Meningoencephalitis: A Case Report. Lancet 2016, 388, 498– 503, DOI: 10.1016/S0140-6736(16)30386-5Google Scholar42Late Ebola virus relapse causing meningoencephalitis: a case reportJacobs Michael; Rodger Alison; Bell David J; MacConnachie Alisdair; Perry Colin; Peters S Erica; Porter Duncan; Ritchie David; Ritchie Neil D; Seaton R Andrew; Bhagani Sanjay; Cropley Ian; Hopkins Susan; Karageorgopoulos Drosos; Lester Rebecca; Mahungu Tabitha; Mepham Stephen; Warren Simon; Filipe Ana; Gifford Robert J; Hughes Joseph; Orton Richard; Palmarini Massimo; Sreenu Vattipally B; Wilkie Gavin S; Jabeen Farrah; Johannessen Ingolfur; Templeton Kate; Lackenby Angie; Zambon Maria; Liu Rebecca S N; Martin Daniel; Marshall Neal; Patel Monika; Gopal Robin; Thomson Emma CLancet (London, England) (2016), 388 (10043), 498-503 ISSN:.BACKGROUND: There are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13.2). METHODS: A 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach. FINDINGS: On admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23.7) than plasma (31.3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus. INTERPRETATION: Our report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern. FUNDING: Royal Free London NHS Foundation Trust.
- 43Schnirring, L. Youngest Ebola Survivor Leaves Guinea Hospital; Center For Infectious Disease Research and Policy, Minneapolis, MN, USA, Nov. 30, 2015; http://www.cidrap.umn.edu/news-perspective/2015/11/youngest-ebola-survivor-leaves-guinea-hospital (accessed Jul. 22, 2016).Google ScholarThere is no corresponding record for this reference.
- 44National Institues of Health. PREVAIL Treatment Trial For Men With Persistent Ebola Viral RNA in Semen Opens in Liberia, Jul. 5, 2016; https://www.nih.gov/news-events/news-releases/prevail-treatment-trial-men-persistent-ebola-viral-rna-semen-opens-liberia (accessed, Jul., 22, 2016).Google ScholarThere is no corresponding record for this reference.
Cited By
Click to copy section linkSection link copied!
This article is cited by 524 publications.
- Dean G. Brown, Hyejin Park. Current and Emerging Prodrug Strategies. Journal of Medicinal Chemistry 2025, 68
(12)
, 12369-12391. https://doi.org/10.1021/acs.jmedchem.5c00826
- Monta Nakamura, Kiyoshi Kakuta, Kazuki Sato, Takeshi Wada. Stereoselective Synthesis of Nucleotide Analog Prodrugs (ProTides) via an Oxazaphospholidine Method. The Journal of Organic Chemistry 2025, 90
(19)
, 6434-6442. https://doi.org/10.1021/acs.joc.5c00240
- Sourav Pal, Quinlin M. Hanson, Sarah C. Ogden, Emily M. Lee, Natalia J. Martinez, Alexey V. Zakharov. Discovery of SARS-CoV-2 Nsp14-Methyltransferase (MTase) Inhibitors by Harnessing Scaffold-Centric Exploration of the Ultra Large Chemical Space. ACS Pharmacology & Translational Science 2025, 8
(5)
, 1366-1400. https://doi.org/10.1021/acsptsci.5c00111
- Aticha Thiraporn, Thanat Tiyasakulchai, Thitiphong Khamkhenshorngphanuch, Marie Hoarau, Ratthiya Thiabma, Suppachoke Onnome, Amporn Suphatrakul, Jaraspim Narkpuk, Chanya Srisaowakarn, Suwimon Manopwisedjaroen, Kitlada Srichomthong, Suradej Hongeng, Arunee Thitithanyanont, Peera Jaru-ampornpan, Sewan Theeramunkong, Bunpote Siridechadilok, Nitipol Srimongkolpithak. Synthesis and Modification of Cordycepin-Phosphoramidate ProTide Derivatives for Antiviral Activity and Metabolic Stability. ACS Bio & Med Chem Au 2025, 5
(1)
, 89-105. https://doi.org/10.1021/acsbiomedchemau.4c00071
- Xin Li, Yongcheng Song. Perspective for Drug Discovery Targeting SARS Coronavirus Methyltransferases: Function, Structure and Inhibition. Journal of Medicinal Chemistry 2024, 67
(21)
, 18642-18655. https://doi.org/10.1021/acs.jmedchem.4c01749
- Thitiphong Khamkhenshorngphanuch, Pitchayathida Mee-udorn, Maleeruk Utsintong, Chutima Thepparit, Nitipol Srimongkolpithak, Sewan Theeramunkong. Study of Hydrolysis Kinetics and Synthesis of Single Isomer of Phosphoramidate ProTide-Acyclovir. ACS Omega 2024, 9
(45)
, 45221-45231. https://doi.org/10.1021/acsomega.4c06645
- Shi-Chao Wang, Lin Liu, Mei Duan, Weijia Xie, Jiabin Han, Yuhang Xue, You Wang, Xiaotai Wang, Shaolin Zhu. Regio- and Enantioselective Nickel-Catalyzed Ipso- and Remote Hydroamination Utilizing Organic Azides as Amino Sources for the Synthesis of Primary Amines. Journal of the American Chemical Society 2024, 146
(44)
, 30626-30636. https://doi.org/10.1021/jacs.4c12324
- Terence C. S. Ho, Alex H. Y. Chan, Finian J. Leeper. Triazole-Based Thiamine Analogues as Inhibitors of Thiamine Pyrophosphate-Dependent Enzymes: 1,3-Dicarboxylate for Metal Binding. ACS Omega 2024, 9
(41)
, 42245-42252. https://doi.org/10.1021/acsomega.4c04594
- Dustin S. Siegel, Hon C. Hui, Jared Pitts, Meghan S. Vermillion, Kazuya Ishida, Davin Rautiola, Michael Keeney, Hammad Irshad, Lijun Zhang, Kwon Chun, Gregory Chin, Bindu Goyal, Edward Doerffler, Hai Yang, Michael O. Clarke, Chris Palmiotti, Arya Vijjapurapu, Nicholas C. Riola, Kirsten Stray, Eisuke Murakami, Bin Ma, Ting Wang, Xiaofeng Zhao, Yili Xu, Gary Lee, Bruno Marchand, Minji Seung, Arabinda Nayak, Adrian Tomkinson, Nani Kadrichu, Scott Ellis, Ona Barauskas, Joy Y. Feng, Jason K. Perry, Michel Perron, John P. Bilello, Philip J. Kuehl, Raju Subramanian, Tomas Cihlar, Richard L. Mackman. Discovery of GS-7682, a Novel 4′-Cyano-Modified C-Nucleoside Prodrug with Broad Activity against Pneumo- and Picornaviruses and Efficacy in RSV-Infected African Green Monkeys. Journal of Medicinal Chemistry 2024, 67
(15)
, 12945-12968. https://doi.org/10.1021/acs.jmedchem.4c00899
- Tommaso Felicetti, Chiara Sarnari, Roberta Gaito, Oriana Tabarrini, Giuseppe Manfroni. Recent Progress toward the Discovery of Small Molecules as Novel Anti-Respiratory Syncytial Virus Agents. Journal of Medicinal Chemistry 2024, 67
(14)
, 11543-11579. https://doi.org/10.1021/acs.jmedchem.4c00630
- Qishu Chen, Qifan Zhou, Guanguan Li, Shuo Li, Yingjun Li, Xumu Zhang. Optimized Kilogram-Scale Synthesis and Impurity Identification of SHEN26 (ATV014) for Treating COVID-19. Organic Process Research & Development 2024, 28
(6)
, 2188-2195. https://doi.org/10.1021/acs.oprd.3c00248
- Kewen Peng, Shamar D. Wallace, Saket R. Bagde, Jialin Shang, Ananya Anmangandla, Sadhan Jana, J. Christopher Fromme, Hening Lin. GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains. ACS Chemical Biology 2024, 19
(5)
, 1093-1105. https://doi.org/10.1021/acschembio.4c00027
- Eugen F. Mesaros, Benjamin J. Dugan, Min Gao, Muhammad Sheraz, Kayleigh McGovern-Gooch, Fran Xu, Kristi Yi Fan, Duyan Nguyen, Steven G. Kultgen, Aaron Lindstrom, Kim Stever, Breanna Tercero, Randall J. Binder, Fei Liu, Holly M. Micolochick Steuer, Nagraj Mani, Troy O. Harasym, Emily P. Thi, Andrea Cuconati, Bruce D. Dorsey, Andrew G. Cole, Angela M. Lam, Michael J. Sofia. Discovery of C-Linked Nucleoside Analogues with Antiviral Activity against SARS-CoV-2. ACS Infectious Diseases 2024, 10
(5)
, 1780-1792. https://doi.org/10.1021/acsinfecdis.4c00122
- Jiapeng Li, Daniel Macedo de Melo Jorge, Weiwen Wang, Shuxin Sun, Tristan Frum, Yu-An Hang, Yueting Liu, Xingwu Zhou, Jingcheng Xiao, Xinwen Wang, Jason R. Spence, Christiane E. Wobus, Hao-Jie Zhu. Differential Bioactivation Profiles of Different GS-441524 Prodrugs in Cell and Mouse Models: ProTide Prodrugs with High Cell Permeability and Susceptibility to Cathepsin A Are More Efficient in Delivering Antiviral Active Metabolites to the Lung. Journal of Medicinal Chemistry 2024, 67
(9)
, 7470-7486. https://doi.org/10.1021/acs.jmedchem.4c00234
- Xue-E Guan, Run-Ping Miao, Xia Hua, Xiao Jin, Guo-Zhong Deng, Bao-Dong Cui, Wen-Yong Han, Nan-Wei Wan, Yong-Zheng Chen. Biocatalytic Enantioselective Synthesis of Chiral β-Hydroxy Nitriles Using Cyanohydrins as Cyano Sources. ACS Catalysis 2023, 13
(20)
, 13597-13606. https://doi.org/10.1021/acscatal.3c03173
- Richard L. Mackman, Rao V. Kalla, Darius Babusis, Jared Pitts, Kimberly T. Barrett, Kwon Chun, Venice Du Pont, Lauren Rodriguez, Jasmine Moshiri, Yili Xu, Michael Lee, Gary Lee, Blake Bleier, Anh-Quan Nguyen, B. Michael O’Keefe, Andrea Ambrosi, Meredith Cook, Joy Yu, Kassibla Elodie Dempah, Elaine Bunyan, Nicholas C. Riola, Xianghan Lu, Renmeng Liu, Ashley Davie, Tien-Ying Hsiang, Justin Dearing, Meghan Vermillion, Michael Gale, Jr., Anita Niedziela-Majka, Joy Y. Feng, Charlotte Hedskog, John P. Bilello, Raju Subramanian, Tomas Cihlar. Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 That Exhibits Antiviral Efficacy in SARS-CoV-2-Infected African Green Monkeys. Journal of Medicinal Chemistry 2023, 66
(17)
, 11701-11717. https://doi.org/10.1021/acs.jmedchem.3c00750
- Marta Gargantilla, Clara Francés, Anmol Adhav, Alicia Forcada-Nadal, Belén Martínez-Gualda, Olaia Martí-Marí, María Luisa López-Redondo, Roberto Melero, Clara Marco-Marín, Nadine Gougeard, Carolina Espinosa, Antonio Rubio-del-Campo, Rafael Ruiz-Partida, María del Pilar Hernández-Sierra, Laura Villamayor-Belinchón, Jerónimo Bravo, José-Luis Llacer, Alberto Marina, Vicente Rubio, Ana San-Félix, Ron Geller, María-Jesús Pérez-Pérez. C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein. Journal of Medicinal Chemistry 2023, 66
(15)
, 10432-10457. https://doi.org/10.1021/acs.jmedchem.3c00576
- Jie Lv, Juan Zou, Yingling Nong, Jia Song, Tingwei Shen, Hui Cai, Chengli Mou, Wenxin Lyu, Zhichao Jin, Yonggui Robin Chi. Catalytic Regioselective Acylation of Unprotected Nucleosides for Quick Access to COVID and Other Nucleoside Prodrugs. ACS Catalysis 2023, 13
(14)
, 9567-9576. https://doi.org/10.1021/acscatal.3c02069
- Savio Cardoza, Manoj Kumar Shrivash, Laura Riva, Arnab K. Chatterjee, Ajay Mandal, Vibha Tandon. Multistep Synthesis of Analogues of Remdesivir: Incorporating Heterocycles at the C-1′ Position. The Journal of Organic Chemistry 2023, 88
(13)
, 9105-9122. https://doi.org/10.1021/acs.joc.3c00754
- Isha Singh, Fengling Li, Elissa A. Fink, Irene Chau, Alice Li, Annía Rodriguez-Hernández, Isabella Glenn, Francisco J. Zapatero-Belinchón, M. Luis Rodriguez, Kanchan Devkota, Zhijie Deng, Kris White, Xiaobo Wan, Nataliya A. Tolmachova, Yurii S. Moroz, H. Ümit Kaniskan, Melanie Ott, Adolfo García-Sastre, Jian Jin, Danica Galonić Fujimori, John J. Irwin, Masoud Vedadi, Brian K. Shoichet. Structure-Based Discovery of Inhibitors of the SARS-CoV-2 Nsp14 N7-Methyltransferase. Journal of Medicinal Chemistry 2023, 66
(12)
, 7785-7803. https://doi.org/10.1021/acs.jmedchem.2c02120
- Ananya Anmangandla, Sadhan Jana, Kewen Peng, Shamar D. Wallace, Saket R. Bagde, Bryon S. Drown, Jiashu Xu, Paul J. Hergenrother, J. Christopher Fromme, Hening Lin. A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain. ACS Chemical Biology 2023, 18
(5)
, 1200-1207. https://doi.org/10.1021/acschembio.3c00092
- Richard L. Mackman*Tomas Cihlar. VEKLURY® (REMDESIVIR), A NUCLEOTIDE PRODRUG APPROVED FOR THE TREATMENT OF COVID-19. , 545-569. https://doi.org/10.1021/mc-2022-vol57.ch22
- Trung Cao Natalia Dyatkina Sébastien Lemaire Marija Prhavc Simon Wagschal . Discovery and Chemical Development of Adafosbuvir, a Nucleoside Phosphoramidate Prodrug for the Treatment of Hepatitis C Infection. , 95-141. https://doi.org/10.1021/bk-2022-1423.ch002
- Marya Y. Ornelas, Angela Y. Thomas, L. Idalee Johnson Rosas, Riley O. Scoville, Angad P. Mehta. Synthetic Platforms for Characterizing and Targeting of SARS-CoV-2 Genome Capping Enzymes. ACS Synthetic Biology 2022, 11
(11)
, 3759-3771. https://doi.org/10.1021/acssynbio.2c00359
- Chao Yang, Lenka Poštová Slavětínská, Marianne Fleuti, Blanka Klepetářová, Michal Tichý, Soňa Gurská, Petr Pavliš, Petr Džubák, Marián Hajdúch, Michal Hocek. Synthesis of Polycyclic Hetero-Fused 7-Deazapurine Heterocycles and Nucleosides through C–H Dibenzothiophenation and Negishi Coupling. Journal of the American Chemical Society 2022, 144
(42)
, 19437-19446. https://doi.org/10.1021/jacs.2c07517
- Hongxiang Hu, Mohamed Dit Mady Traore, Ruiting Li, Hebao Yuan, Miao He, Bo Wen, Wei Gao, Colleen B. Jonsson, Elizabeth A. Fitzpatrick, Duxin Sun. Optimization of the Prodrug Moiety of Remdesivir to Improve Lung Exposure/Selectivity and Enhance Anti-SARS-CoV-2 Activity. Journal of Medicinal Chemistry 2022, 65
(18)
, 12044-12054. https://doi.org/10.1021/acs.jmedchem.2c00758
- Mo Wang, Zhenfeng Zhang, Wanbin Zhang. Design, Synthesis, and Application of Chiral Bicyclic Imidazole Catalysts. Accounts of Chemical Research 2022, 55
(18)
, 2708-2727. https://doi.org/10.1021/acs.accounts.2c00455
- Severino Jefferson Ribeiro da
Silva, Jessica Catarine Frutuoso do Nascimento, Renata Pessôa Germano Mendes, Klarissa Miranda Guarines, Caroline Targino Alves da Silva, Poliana Gomes da Silva, Jurandy Júnior Ferraz de Magalhães, Justin R. J. Vigar, Abelardo Silva-Júnior, Alain Kohl, Keith Pardee, Lindomar Pena. Two Years into the COVID-19 Pandemic: Lessons Learned. ACS Infectious Diseases 2022, 8
(9)
, 1758-1814. https://doi.org/10.1021/acsinfecdis.2c00204
- Andrew C. Flick, Carolyn A. Leverett, Hong X. Ding, Emma L. McInturff, Sarah J. Fink, Subham Mahapatra, Daniel W. Carney, Erick A. Lindsey, Jacob C. DeForest, Scott P. France, Simon Berritt, Simone V. Bigi-Botterill, Tony S. Gibson, Rebecca B. Watson, Yiyang Liu, Christopher J. O’Donnell. Synthetic Approaches to the New Drugs Approved During 2020. Journal of Medicinal Chemistry 2022, 65
(14)
, 9607-9661. https://doi.org/10.1021/acs.jmedchem.2c00710
- Sehr Naseem-Khan, Madison B. Berger, Emmett M. Leddin, Yazdan Maghsoud, G. Andrés Cisneros. Impact of Remdesivir Incorporation along the Primer Strand on SARS-CoV-2 RNA-Dependent RNA Polymerase. Journal of Chemical Information and Modeling 2022, 62
(10)
, 2456-2465. https://doi.org/10.1021/acs.jcim.2c00201
- Prashant Khirsariya, Patrik Pospíšil, Lukáš Maier, Miroslav Boudný, Martin Babáš, Ondřej Kroutil, Marek Mráz, Robert Vácha, Kamil Paruch. Synthesis and Profiling of Highly Selective Inhibitors of Methyltransferase DOT1L Based on Carbocyclic C-Nucleosides. Journal of Medicinal Chemistry 2022, 65
(7)
, 5701-5723. https://doi.org/10.1021/acs.jmedchem.1c02228
- Andrew C. Stevens, Katrien Brak, W. Stacy Bremner, Angela M. Brown, Andrei Chtchemelinine, Lars Heumann, James A. Kerschen, Witold Subotkowski, Tiago Vieira, Lydia C. Wolfe, Boran Xu, Chia-Yun Yu. Development of a Scalable Lanthanide Halide/Quaternary Ammonium Salt System for the Nucleophilic Addition of Grignard Reagents to Carbonyl Groups and Application to the Synthesis of a Remdesivir Intermediate. Organic Process Research & Development 2022, 26
(3)
, 754-763. https://doi.org/10.1021/acs.oprd.1c00191
- Greg T. Cizio, Kathy Dao, Edward M. Doerffler, Nolan D. Griggs, Michael A. Ischay, Baldip S. Kang, Matthew M. Logan, Patricia D. MacLeod, Adam B. Weinstein, Lok Him L. Yu. Early Development and Kilogram Scale-Up of a Non-steroidal FXR Agonist for the Treatment of Non-alcoholic Steatohepatitis (NASH). Organic Process Research & Development 2022, 26
(3)
, 745-753. https://doi.org/10.1021/acs.oprd.1c00193
- Richard L. Mackman. Phosphoramidate Prodrugs Continue to Deliver, The Journey of Remdesivir (GS-5734) from RSV to SARS-CoV-2. ACS Medicinal Chemistry Letters 2022, 13
(3)
, 338-347. https://doi.org/10.1021/acsmedchemlett.1c00624
- Rolando Cannalire, Carmen Cerchia, Andrea R. Beccari, Francesco Saverio Di Leva, Vincenzo Summa. Targeting SARS-CoV-2 Proteases and Polymerase for COVID-19 Treatment: State of the Art and Future Opportunities. Journal of Medicinal Chemistry 2022, 65
(4)
, 2716-2746. https://doi.org/10.1021/acs.jmedchem.0c01140
- Yingjun Li, Liu Cao, Ge Li, Feng Cong, Yunfeng Li, Jing Sun, Yinzhu Luo, Guijiang Chen, Guanguan Li, Ping Wang, Fan Xing, Yanxi Ji, Jincun Zhao, Yu Zhang, Deyin Guo, Xumu Zhang. Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models. Journal of Medicinal Chemistry 2022, 65
(4)
, 2785-2793. https://doi.org/10.1021/acs.jmedchem.0c01929
- Sarabindu Roy, Ajay Yadaw, Subho Roy, Gopal Sirasani, Aravind Gangu, Jack D. Brown, Joseph D. Armstrong, III, Rodger W. Stringham, B. Frank Gupton, Chris H. Senanayake, David R. Snead. Facile and Scalable Methodology for the Pyrrolo[2,1-f][1,2,4]triazine of Remdesivir. Organic Process Research & Development 2022, 26
(1)
, 82-90. https://doi.org/10.1021/acs.oprd.1c00071
- Jun-Qi Zhang, Jiayue Liu, Dandan Hu, Jinyu Song, Guorong Zhu, Hongjun Ren. Rapid and Simple Access to α-(Hetero)arylacetonitriles from Gem-Difluoroalkenes. Organic Letters 2022, 24
(2)
, 786-790. https://doi.org/10.1021/acs.orglett.1c04336
- Subhankar Panda, Tej Narayan Poudel, Pooja Hegde, Courtney C. Aldrich. Innovative Strategies for the Construction of Diverse 1′-Modified C-Nucleoside Derivatives. The Journal of Organic Chemistry 2021, 86
(23)
, 16625-16640. https://doi.org/10.1021/acs.joc.1c01920
- Youngran Seo, Jared M. Lowe, Neyen Romano, Michel R. Gagné. Switching between X-Pyrano-, X-Furano-, and Anhydro-X-pyranoside Synthesis (X = C, N) under Lewis acid Catalyzed Conditions. Organic Letters 2021, 23
(15)
, 5636-5640. https://doi.org/10.1021/acs.orglett.1c01713
- Didier F. Vargas, Enrique L. Larghi, Teodoro S. Kaufman. Evolution of the Synthesis of Remdesivir. Classical Approaches and Most Recent Advances. ACS Omega 2021, 6
(30)
, 19356-19363. https://doi.org/10.1021/acsomega.1c03082
- Violaine Desgens-Martin, Arturo A. Keller. COVID-19 Treatment Agents: Do They Pose an Environmental Risk?. ACS ES&T Water 2021, 1
(7)
, 1555-1565. https://doi.org/10.1021/acsestwater.1c00059
- David L. Hughes. Quest for a Cure: Potential Small-Molecule Treatments for COVID-19, Part 2. Organic Process Research & Development 2021, 25
(5)
, 1089-1111. https://doi.org/10.1021/acs.oprd.1c00100
- Richard L. Mackman, Hon C. Hui, Michel Perron, Eisuke Murakami, Christopher Palmiotti, Gary Lee, Kirsten Stray, Lijun Zhang, Bindu Goyal, Kwon Chun, Daniel Byun, Dustin Siegel, Scott Simonovich, Venice Du Pont, Jared Pitts, Darius Babusis, Arya Vijjapurapu, Xianghan Lu, Cynthia Kim, Xiaofeng Zhao, Julie Chan, Bin Ma, Diane Lye, Adelle Vandersteen, Sarah Wortman, Kimberly T. Barrett, Maria Toteva, Robert Jordan, Raju Subramanian, John P. Bilello, Tomas Cihlar. Prodrugs of a 1′-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV. Journal of Medicinal Chemistry 2021, 64
(8)
, 5001-5017. https://doi.org/10.1021/acs.jmedchem.1c00071
- Timo von Keutz, Jason D. Williams, C. Oliver Kappe. Flash Chemistry Approach to Organometallic C-Glycosylation for the Synthesis of Remdesivir. Organic Process Research & Development 2021, 25
(4)
, 1015-1021. https://doi.org/10.1021/acs.oprd.1c00024
- Jiapeng Li, Shuhan Liu, Jian Shi, Xinwen Wang, Yanling Xue, Hao-Jie Zhu. Tissue-Specific Proteomics Analysis of Anti-COVID-19 Nucleoside and Nucleotide Prodrug-Activating Enzymes Provides Insights into the Optimization of Prodrug Design and Pharmacotherapy Strategy. ACS Pharmacology & Translational Science 2021, 4
(2)
, 870-887. https://doi.org/10.1021/acsptsci.1c00016
- Ting Chen, Cheng-Yin Fei, Yi-Ping Chen, Karen Sargsyan, Chun-Ping Chang, Hanna S. Yuan, Carmay Lim. Synergistic Inhibition of SARS-CoV-2 Replication Using Disulfiram/Ebselen and Remdesivir. ACS Pharmacology & Translational Science 2021, 4
(2)
, 898-907. https://doi.org/10.1021/acsptsci.1c00022
- Ei-ichi Ami, Hiroshi Ohrui. Intriguing Antiviral Modified Nucleosides: A Retrospective View into the Future Treatment of COVID-19. ACS Medicinal Chemistry Letters 2021, 12
(4)
, 510-517. https://doi.org/10.1021/acsmedchemlett.1c00070
- Veeranjaneyulu Gannedi, Bharath Kumar Villuri, Sivakumar N. Reddy, Chiao-Chu Ku, Chi-Huey Wong, Shang-Cheng Hung. Practical Remdesivir Synthesis through One-Pot Organocatalyzed Asymmetric (S)-P-Phosphoramidation. The Journal of Organic Chemistry 2021, 86
(7)
, 4977-4985. https://doi.org/10.1021/acs.joc.0c02888
- Yuanchao Xie, Tianwen Hu, Yan Zhang, Daibao Wei, Wei Zheng, Fuqiang Zhu, Guanghui Tian, Haji A. Aisa, Jingshan Shen. Weinreb Amide Approach to the Practical Synthesis of a Key Remdesivir Intermediate. The Journal of Organic Chemistry 2021, 86
(7)
, 5065-5072. https://doi.org/10.1021/acs.joc.0c02986
- Andrew J. Prussia, Spandan Chennamadhavuni. Biostructural Models for the Binding of Nucleoside Analogs to SARS-CoV-2 RNA-Dependent RNA Polymerase. Journal of Chemical Information and Modeling 2021, 61
(3)
, 1402-1411. https://doi.org/10.1021/acs.jcim.0c01277
- Nemanja Milisavljevic, Eva Konkolová, Jaroslav Kozák, Jan Hodek, Lucia Veselovská, Veronika Sýkorová, Karel Čížek, Radek Pohl, Luděk Eyer, Pavel Svoboda, Daniel Růžek, Jan Weber, Radim Nencka, Evžen Bouřa, Michal Hocek. Antiviral Activity of 7-Substituted 7-Deazapurine Ribonucleosides, Monophosphate Prodrugs, and Triphoshates against Emerging RNA Viruses. ACS Infectious Diseases 2021, 7
(2)
, 471-478. https://doi.org/10.1021/acsinfecdis.0c00829
- Yi Zhang, Liang V. Tang. Overview of Targets and Potential Drugs of SARS-CoV-2 According to the Viral Replication. Journal of Proteome Research 2021, 20
(1)
, 49-59. https://doi.org/10.1021/acs.jproteome.0c00526
- Sara S. Rocks Robert A. Stockland , Jr. . Heteronuclear NMR Spectroscopy in the Undergraduate Curriculum: Direct and Indirect Effects. 2021, 191-208. https://doi.org/10.1021/bk-2021-1376.ch013
- Eugene Mamontov, Yongqiang Cheng, Luke L. Daemen, Alexander I. Kolesnikov, Anibal J. Ramirez-Cuesta, Matthew R. Ryder, Matthew B. Stone. Hydration-Induced Disorder Lowers the Energy Barriers for Methyl Rotation in Drug Molecules. The Journal of Physical Chemistry Letters 2020, 11
(23)
, 10256-10261. https://doi.org/10.1021/acs.jpclett.0c02642
- Padmaja D. Wakchaure, Shibaji Ghosh, Bishwajit Ganguly. Revealing the Inhibition Mechanism of RNA-Dependent RNA Polymerase (RdRp) of SARS-CoV-2 by Remdesivir and Nucleotide Analogues: A Molecular Dynamics Simulation Study. The Journal of Physical Chemistry B 2020, 124
(47)
, 10641-10652. https://doi.org/10.1021/acs.jpcb.0c06747
- Marwa O. Mikati, Justin J. Miller, Damon M. Osbourn, Yasaman Barekatain, Naomi Ghebremichael, Ishaan T. Shah, Carey-Ann D. Burnham, Kenneth M. Heidel, Victoria C. Yan, Florian L. Muller, Cynthia S. Dowd, Rachel L. Edwards, Audrey R. Odom John. Antimicrobial Prodrug Activation by the Staphylococcal Glyoxalase GloB. ACS Infectious Diseases 2020, 6
(11)
, 3064-3075. https://doi.org/10.1021/acsinfecdis.0c00582
- Rachel R. Knapp, Veronica Tona, Taku Okada, Richmond Sarpong, Neil K. Garg. Cyanoamidine Cyclization Approach to Remdesivir’s Nucleobase. Organic Letters 2020, 22
(21)
, 8430-8435. https://doi.org/10.1021/acs.orglett.0c03052
- Xiao Jia, Stefan Weber, Dominique Schols, Chris Meier. Membrane Permeable, Bioreversibly Modified Prodrugs of Nucleoside Diphosphate-γ-Phosphonates. Journal of Medicinal Chemistry 2020, 63
(20)
, 11990-12007. https://doi.org/10.1021/acs.jmedchem.0c01294
- Tiago Vieira, Andrew C. Stevens, Andrei Chtchemelinine, Detian Gao, Pavel Badalov, Lars Heumann. Development of a Large-Scale Cyanation Process Using Continuous Flow Chemistry En Route to the Synthesis of Remdesivir. Organic Process Research & Development 2020, 24
(10)
, 2113-2121. https://doi.org/10.1021/acs.oprd.0c00172
- Timo von Keutz, Jason D. Williams, C. Oliver Kappe. Continuous Flow C-Glycosylation via Metal–Halogen Exchange: Process Understanding and Improvements toward Efficient Manufacturing of Remdesivir. Organic Process Research & Development 2020, 24
(10)
, 2362-2368. https://doi.org/10.1021/acs.oprd.0c00370
- Matthew D. Lloyd. High-Throughput Screening for the Discovery of Enzyme Inhibitors. Journal of Medicinal Chemistry 2020, 63
(19)
, 10742-10772. https://doi.org/10.1021/acs.jmedchem.0c00523
- Dinesh J. Paymode, Flavio S. P. Cardoso, Toolika Agrawal, John W. Tomlin, Daniel W. Cook, Justina M. Burns, Rodger W. Stringham, Joshua D. Sieber, B. Frank Gupton, David R. Snead. Expanding Access to Remdesivir via an Improved Pyrrolotriazine Synthesis: Supply Centered Synthesis. Organic Letters 2020, 22
(19)
, 7656-7661. https://doi.org/10.1021/acs.orglett.0c02848
- Ilya G. Shenderovich. For Whom a Puddle Is the Sea? Adsorption of Organic Guests on Hydrated MCM-41 Silica. Langmuir 2020, 36
(38)
, 11383-11392. https://doi.org/10.1021/acs.langmuir.0c02327
- Fei Xue, Xinbo Zhou, Ruijie Zhou, Xiaohan Zhou, Dian Xiao, Eric Gu, Xiaowen Guo, Ji Xiang, Ke Wang, Likai Yang, Wu Zhong, Yong Qin. Improvement of the C-glycosylation Step for the Synthesis of Remdesivir. Organic Process Research & Development 2020, 24
(9)
, 1772-1777. https://doi.org/10.1021/acs.oprd.0c00310
- Andrew N. Bigley, Tamari Narindoshvili, Frank M. Raushel. A Chemoenzymatic Synthesis of the (RP)-Isomer of the Antiviral Prodrug Remdesivir. Biochemistry 2020, 59
(33)
, 3038-3043. https://doi.org/10.1021/acs.biochem.0c00591
- Andrew J. Wiemer. Metabolic Efficacy of Phosphate Prodrugs and the Remdesivir Paradigm. ACS Pharmacology & Translational Science 2020, 3
(4)
, 613-626. https://doi.org/10.1021/acsptsci.0c00076
- Qingfeng Li, Elisabetta Groaz, Leentje Persoons, Dirk Daelemans, Piet Herdewijn. Synthesis and Antitumor Activity of C-7-Alkynylated and Arylated Pyrrolotriazine C-Ribonucleosides. ACS Medicinal Chemistry Letters 2020, 11
(8)
, 1605-1610. https://doi.org/10.1021/acsmedchemlett.0c00269
- Melissa A. Hardy, Brandon A. Wright, J. Logan Bachman, Timothy B. Boit, Hannah M. S. Haley, Rachel R. Knapp, Robert F. Lusi, Taku Okada, Veronica Tona, Neil K. Garg, Richmond Sarpong. Treating a Global Health Crisis with a Dose of Synthetic Chemistry. ACS Central Science 2020, 6
(7)
, 1017-1030. https://doi.org/10.1021/acscentsci.0c00637
- Wen-Biao Wu, Jin-Sheng Yu, Jian Zhou. Catalytic Enantioselective Cyanation: Recent Advances and Perspectives. ACS Catalysis 2020, 10
(14)
, 7668-7690. https://doi.org/10.1021/acscatal.0c01918
- Peter Finkbeiner, Jörg P. Hehn, Christian Gnamm. Phosphine Oxides from a Medicinal Chemist’s Perspective: Physicochemical and in Vitro Parameters Relevant for Drug Discovery. Journal of Medicinal Chemistry 2020, 63
(13)
, 7081-7107. https://doi.org/10.1021/acs.jmedchem.0c00407
- Victoria C. Yan, Florian L. Muller. Advantages of the Parent Nucleoside GS-441524 over Remdesivir for Covid-19 Treatment. ACS Medicinal Chemistry Letters 2020, 11
(7)
, 1361-1366. https://doi.org/10.1021/acsmedchemlett.0c00316
- Chris De Savi, David L. Hughes, Lisbet Kvaerno. Quest for a COVID-19 Cure by Repurposing Small-Molecule Drugs: Mechanism of Action, Clinical Development, Synthesis at Scale, and Outlook for Supply. Organic Process Research & Development 2020, 24
(6)
, 940-976. https://doi.org/10.1021/acs.oprd.0c00233
- Xiao Jia, Dominique Schols, Chris Meier. Anti-HIV-Active Nucleoside Triphosphate Prodrugs. Journal of Medicinal Chemistry 2020, 63
(11)
, 6003-6027. https://doi.org/10.1021/acs.jmedchem.0c00271
- Richard T. Eastman, Jacob S. Roth, Kyle R. Brimacombe, Anton Simeonov, Min Shen, Samarjit Patnaik, Matthew D. Hall. Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19. ACS Central Science 2020, 6
(5)
, 672-683. https://doi.org/10.1021/acscentsci.0c00489
- Haidi Li, Jie Chao, Jaafar Hasan, Guang Tian, Yatao Jin, Zixin Zhang, Chuanguang Qin. Synthesis of Tri(4-formylphenyl) Phosphonate Derivatives as Recyclable Triple-Equivalent Supports of Peptide Synthesis. The Journal of Organic Chemistry 2020, 85
(10)
, 6271-6280. https://doi.org/10.1021/acs.joc.9b03023
- Dean G. Brown, Jonas Boström. Where Do Recent Small Molecule Clinical Development Candidates Come From?. Journal of Medicinal Chemistry 2018, 61
(21)
, 9442-9468. https://doi.org/10.1021/acs.jmedchem.8b00675
- Ke-Xin Huang, Ming-Sheng Xie, Qi-Ying Zhang, Gui-Rong Qu, and Hai-Ming Guo . Enantioselective Synthesis of Carbocyclic Nucleosides via Asymmetric [3 + 2] Annulation of α-Purine-Substituted Acrylates with MBH Carbonates. Organic Letters 2018, 20
(2)
, 389-392. https://doi.org/10.1021/acs.orglett.7b03625
- Wanqing Wu, Zhiming Lin, Chuanle Zhu, Pengquan Chen, Jiawei Li, and Huanfeng Jiang . Transition-Metal-Free [3+2] Cycloaddition of Dehydroaminophosphonates and N-Tosylhydrazones: Access to Aminocyclopropanephosphonates with Adjacent Quaternary-Tetrasubstituted Carbon Centers. The Journal of Organic Chemistry 2017, 82
(23)
, 12746-12756. https://doi.org/10.1021/acs.joc.7b01862
- Hubert Hřebabecký, Martin Dračínský, Eliška Procházková, Michal Šála, Richard Mackman, and Radim Nencka . Control of α/β Anomer Formation by a 2′,5′ Bridge: Toward Nucleoside Derivatives Locked in the South Conformation. The Journal of Organic Chemistry 2017, 82
(21)
, 11337-11347. https://doi.org/10.1021/acs.joc.7b01000
- Maria Giulia Nizi, Serena Massari, Oriana Tabarrini, Giuseppe Manfroni. A medicinal chemistry overview of direct-acting antivirals approved in 2013–2024 by US FDA. European Journal of Medicinal Chemistry 2025, 300 , 118105. https://doi.org/10.1016/j.ejmech.2025.118105
- A Ram Lee, Hong Ki Min, Seon-Yeong Lee, Su Been Jeon, Chae Rim Lee, Tae Ho Kim, Jin Hyung Park, Mi- La Cho. Remdesivir alleviates joint damage in collagen-induced arthritis and inhibits inflammatory cell death of RA synovial fibroblasts. Immunology Letters 2025, 275 , 107009. https://doi.org/10.1016/j.imlet.2025.107009
- Sayak Das, Nagendra Thakur, Arpita S. Harnam, Janusz T. Paweska, Ahmed S. Abdel-Moneim, Shailendra K. Saxena. Marburg virus outbreak: a global health threat, zoonotic risks, and the urgent need for international action. VirusDisease 2025, 9 https://doi.org/10.1007/s13337-025-00940-y
- Fangfang Zhu, Xiaozhu Wu, De Wang. Construction of functionalized adjacent P(
v
)–C chiral stereogenic centers
via
organophosphine-catalyzed asymmetric S
N
2′ substitution of unsymmetrical phosphine oxides with MBH adducts. Organic & Biomolecular Chemistry 2025, 23
(36)
, 8186-8192. https://doi.org/10.1039/D5OB01071C
- Oliver J. Read, Jennifer Bré, Ying Zhang, Peter Mullen, Mustafa Elshani, David J. Harrison, . A phosphoramidate modification of FUDR, NUC-3373, causes DNA damage and DAMPs release from colorectal cancer cells, potentiating lymphocyte-induced cell death. PLOS One 2025, 20
(9)
, e0331567. https://doi.org/10.1371/journal.pone.0331567
- Andrew N. Bigley, Frank M. Raushel. The use of phosphotriesterase in the synthesis of enantiomerically pure ProTide prodrugs. Chemico-Biological Interactions 2025, 418 , 111597. https://doi.org/10.1016/j.cbi.2025.111597
- Laura Vandemaele, Thibault Francken, Joost Schepers, Winston Chiu, Niels Cremers, Hugo Klaassen, Charlène Marcadet, Lorena Sanchez Felipe, Arnaud Marchand, Patrick Chaltin, Pieter Leyssen, Johan Neyts, Manon Laporte. High-throughput split-GFP antiviral screening assay against fusogenic paramyxoviruses. Antiviral Research 2025, 241 , 106242. https://doi.org/10.1016/j.antiviral.2025.106242
- Arka Das, Rana Saha, Kaushal Naithani, Krutika Sonar, Christine Jonathan, Subhendu Bhowmik. 5′‐O‐P‐N Linkages of Phosphoramidate Nucleotides: Chemical Construction and Biological Impact. The Chemical Record 2025, 25
(8)
https://doi.org/10.1002/tcr.202500056
- Admir Salihovic, Andrea Taladriz-Sender, Glenn A. Burley. Preparation of nucleoside analogues: opportunities for innovation at the interface of synthetic chemistry and biocatalysis. Chemical Science 2025, 16
(26)
, 11700-11710. https://doi.org/10.1039/D5SC03026A
- Ghazal Kakavand, Somayeh Arabzadeh, Sohameh Mohebbi, Kayvan Saeedfar, Atefeh Abedini, Masoud Mardani. Impact of remdesivir treatment on factor VIII gene expression and hematological parameters in COVID-19 patients. Microbial Pathogenesis 2025, 204 , 107536. https://doi.org/10.1016/j.micpath.2025.107536
- Darius Babusis, Cynthia Kim, Jesse Yang, Xiaofeng Zhao, Guoju Geng, Carmen Ip, Nathan Kozon, Hoa Le, Jennifer Leung, Jared Pitts, Dustin S. Siegel, Rao Kalla, Bernard Murray, John P. Bilello, Roy Bannister, Richard L. Mackman, Raju Subramanian. Pharmacokinetics and Metabolism of Broad-Spectrum Antivirals Remdesivir and Obeldesivir with a Consideration to Metabolite GS-441524: Same, Similar, or Different?. Viruses 2025, 17
(6)
, 836. https://doi.org/10.3390/v17060836
- Darren M. Roberts, Xin Liu, Suzanne L. Parker, Andrew Burke, Jenny Peek, Jane E. Carland, Bridin Murnion, Vincent Seah, Steven C. Wallis, Chandra D. Sumi, Saurabh Pandey, Hergen Buscher, Anthony Byrne, Indy Sandaradura, David Bowen, Simon Holz, Adam G. Stewart, Krispin M. Hajkowicz, Jason A. Roberts. Population Pharmacokinetic Modelling of Remdesivir and Its Metabolite GS-441524 in Hospitalised Patients with COVID-19. Clinical Pharmacokinetics 2025, 64
(5)
, 743-756. https://doi.org/10.1007/s40262-025-01496-2
- . Remdesivir for the Treatment of COVID‐19 and Other Viral Infections with Pandemic Potential. 2025, 333-364. https://doi.org/10.1002/9783527845088.ch10
- . Discovery and Development of VV116: A Novel Oral Nucleoside Anti‐SARS‐CoV‐2 Drug. 2025, 389-410. https://doi.org/10.1002/9783527845088.ch12
- Weerapha Panatdasirisuk, Suthathip Phetlum, Thanat Tiyasakulchai, Nitipol Srimongkolpithak, Tanaporn Uengwetwanit, Nongluck Jaito. Immobilized Phosphotriesterase as an Enzymatic Resolution for Sofosbuvir Precursor. Catalysts 2025, 15
(4)
, 339. https://doi.org/10.3390/catal15040339
- Noriko Saito-Tarashima, Takaaki Koma, Naoto Hinotani, Keigo Yoshida, Moka Ogasa, Akiho Murai, Syuya Inoue, Tomoyuki Kondo, Naoya Doi, Koichi Tsuneyama, Masako Nomaguchi, Noriaki Minakawa. 3-Deazaguanosine inhibits SARS-CoV-2 viral replication and reduces the risk of COVID-19 pneumonia in hamster. iScience 2025, 28
(4)
, 112140. https://doi.org/10.1016/j.isci.2025.112140
- Shengjie Zhang, Sunggyeol Jeong, Botao Jiang, Harvey Ho. Pharmacokinetic simulations for remdesivir and its metabolites in healthy subjects and patients with renal impairment. Frontiers in Pharmacology 2025, 16 https://doi.org/10.3389/fphar.2025.1488961
- Guo-Sheng Wang, Yu-Xin Shang, Jia-Qi Liu, Ting-Ting Xu. Selective oxidation of tetrahydrofurfuryl alcohol to (S)-5-(hydroxymethyl)dihydrofuran-2(3H)-one by Ag-CeOx/γ-Al2O3 catalyst. Monatshefte für Chemie - Chemical Monthly 2025, 156
(2)
, 183-191. https://doi.org/10.1007/s00706-024-03274-5
- Arturo Oliver-Guimera, Brian G. Murphy, M. Kevin Keel. The Nucleoside Analog GS-441524 Effectively Attenuates the In Vitro Replication of Multiple Lineages of Circulating Canine Distemper Viruses Isolated from Wild North American Carnivores. Viruses 2025, 17
(2)
, 150. https://doi.org/10.3390/v17020150
- Santosh K. Rath, Ashutosh K. Dash, Nandan Sarkar, Mitali Panchpuri. A Glimpse for the subsistence from pandemic SARS-CoV-2 infection. Bioorganic Chemistry 2025, 154 , 107977. https://doi.org/10.1016/j.bioorg.2024.107977
Learn about these metrics
Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.
Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.
The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.
Recommended Articles
Abstract
Figure 1
Figure 1. Structures of antiviral nucleosides and nucleoside phosphonates.
Figure 2
Figure 2. (a) Compound 4tp modeled into the EBOV polymerase active site. Residue Y636 is highlighted in green surface, sits below the ribose, and corresponds to F704 in RSV. Residue E709 is highlighted in red surface, sits in proximity to the 2′-β-H position of the ribose, and corresponds to S282 in HCV. (b) Compound 13tp modeled into the EBOV polymerase active site. The 2′-β-methyl overlaps with residue E709 highlighted in red. (c) Compound 13tp modeled into the HCV polymerase active site. Residue S282 is highlighted in the yellow surface, and the 2′-β-methyl can be accommodated.
Scheme 1
Scheme 1. First Generation Synthesis of 4baaReagents and conditions: (a) n-BuLi, (TMS)Cl, THF, – 78 °C, 25%; (b) 1,2-bis(chlorodimethylsilyl)ethane, NaH, n-BuLi, THF, – 78 °C, 60%; (c) (TMS)CN, BF3·Et2O, CH2Cl2, – 78 °C, 58% (89:11β-17/α); (d) BCl3, CH2Cl2, – 78 °C, 74%; (e) 19, NMI, OP(OMe)3, 21%; (f) OP(OPh)Cl2, Et3N, CH2Cl2, 0 °C, 23%.
Scheme 2
Scheme 2. Second Generation Synthesis of 4baaReagents and conditions: (a) TMSCl, PhMgCl, i-PrMgCl·LiCl, THF, – 20 °C, 40%; (b) TMSCN, TfOH, TMSOTf, CH2Cl2, – 78 °C, 85%; (c) BCl3, CH2Cl2, – 20 °C, 86%; (d) 2,2-dimethoxypropane, H2SO4, acetone, rt, 90%; (e) 22b, MgCl2, (i-Pr)2NEt, MeCN, 50 °C, 70%; (f) 37% HCl, THF, rt, 69%; (g) OP(OPh)Cl2, Et3N, CH2Cl2, – 78 °C, then 4-nitrophenol, Et3N, 0 °C, 80%; (h) i-Pr2O, 39%.
Figure 3
Figure 3. Thermal ellipsoid representations of (a) 22b and (b) 4b.
Scheme 3
Scheme 3. Prodrug SynthesisaProdrug is an undetermined mixture of diastereoisomers unless otherwise indicated.
baa = amino acid, Ala = alanine, Phe = phenylalanine, AIB = 2-aminoisobutyrate, c-Bu = cyclobutyl, c-Pent = cyclopentyl, Pent = pentyl, Neopent = neopentyl, 2-EtBu = 2-ethylbutyl, PNP = p-nitrophenolate, and PFP = pentafluorophenolate.
cLG = leaving group.
dSingle Sp isomer.
eReagent was a single unassigned isomer at phosphorus.
fNA = not applicable.
Figure 4
Figure 4. Concentration–time profiles following 10 mg/kg iv single dose slow bolus administration of 4b in Rhesus (mean ± SD, n = 3 per time point). (a) Plasma profile of prodrug 4b (black circle) and parent nucleoside 4 (blue triangle). (b) Intracellular concentration of active metabolite 4tp in PBMCs (green diamond) and estimated 4tp EBOV IC50 = 5 μM (dashed black line).
References
This article references 44 other publications.
- 1World Health Organization. Ebola Situation Report—10 June 2016. http://apps.who.int/iris/bitstream/10665/208883/1/ebolasitrep_10Jun2016_eng.pdf?ua=1 (accessed Jul. 22, 2016).There is no corresponding record for this reference.
- 2World Health Organization. Ebola Data and Statistics—11 May 2016. http://apps.who.int/gho/data/view.ebola-sitrep.ebola-summary-20160511?lang=en (accessed Jul. 22, 2016).There is no corresponding record for this reference.
- 3Vetter, P.; Fischer, W. A., II; Schibler, M.; Jacobs, M.; Bausch, D. G.; Kaiser, L. Ebola Virus Shedding and Transmission: Review of Current Evidence. J. Infect. Dis. 2016, 214, S177– S184, DOI: 10.1093/infdis/jiw2543Ebola virus shedding and transmission: review of current evidenceVetter, Pauline; Fischer, William A., II; Schibler, Manuel; Jacobs, Michael; Bausch, Daniel G.; Kaiser, LaurentJournal of Infectious Diseases (2016), 214 (Suppl. 3), S177-S184CODEN: JIDIAQ; ISSN:0022-1899. (Oxford University Press)Background. The magnitude of the 2013-2016 Ebola virus disease outbreak in West Africa was unprecedented, with >28 500 reported cases and >11 000 deaths. Understanding the key elements of Ebola virus transmission is necessary to implement adequate infection prevention and control measures to protect healthcare workers and halt transmission in the community. Methods. We performed an extensive PubMed literature review encompassing the period from discovery of Ebola virus, in 1976, until 1 June 2016 to evaluate the evidence on modes of Ebola virus shedding and transmission. Findings. Ebola virus has been isolated by cell culture from blood, saliva, urine, aq. humor, semen, and breast milk from infected or convalescent patients. Ebola virus RNA has been noted in the following body fluids days or months after onset of illness: saliva (22 days), conjunctiva/tears (28 days), stool (29 days), vaginal fluid (33 days), sweat (44 days), urine (64 days), amniotic fluid (38 days), aq. humor (101 days), cerebrospinal fluid (9 mo), breast milk (16 mo [preliminary data[), and semen (18 mo). Nevertheless, the only documented cases of secondary transmission from recovered patients have been through sexual transmission. We did not find strong evidence supporting respiratory or fomite-assocd. transmission.
- 4Mate, S. E.; Kugelman, J. R.; Nyenswah, T. G.; Ladner, J. T.; Wiley, M. R.; Cordier-Lassalle, T.; Christie, A.; Schroth, G. P.; Gross, S. M.; Davies-Wayne, G. J.; Shinde, S. A.; Murugan, R.; Sieh, S. B.; Badio, M.; Fakoli, L.; Taweh, F.; de Wit, E.; van Doremalen, N.; Munster, V. J.; Pettitt, J.; Prieto, K.; Humrighouse, B. W.; Ströher, U.; DiClaro, J. W.; Hensley, L. E.; Schoepp, R. J.; Safronetz, D.; Fair, J.; Kuhn, J. H.; Blackley, D. J.; Laney, A. S.; Williams, D. E.; Lo, T.; Gasasira, A.; Nichol, S. T.; Formenty, P.; Kateh, F. N.; De Cock, K. M.; Bolay, F.; Sanchez-Lockhart, M.; Palacios, G. Molecular Evidence of Sexual Transmission of Ebola Virus. N. Engl. J. Med. 2015, 373, 2448– 2454, DOI: 10.1056/NEJMoa15097734Molecular evidence of sexual transmission of Ebola virusMate, S. E.; Kugelman, J. R.; Nyenswah, T. G.; Ladner, J. T.; Wiley, M. R.; Cordier-Lassalle, T.; Christie, A.; Schroth, G. P.; Gross, S. M.; Davies-Wayne, G. J.; Shinde, S. A.; Murugan, R.; Sieh, S. B.; Badio, M.; Fakoli, L.; Taweh, F.; de Wit, E.; van Doremalen, N.; Munster, V. J.; Pettitt, J.; Prieto, K.; Humrighouse, B. W.; Stroher, U.; Di Claro, J. W.; Hensley, L. E.; Schoepp, R. J.; Safronetz, D.; Fair, J.; Kuhn, J. H.; Blackley, D. J.; Laney, A. S.; Williams, D. E.; Lo, T.; Gasasira, A.; Nichol, S. T.; Formenty, P.; Kateh, F. N.; De Cock, K. M.; Bolay, F.; Sanchez-Lockhart, M.; Palacios, G.New England Journal of Medicine (2015), 373 (25), 2448-2454CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiol. data, the genomic anal. provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD.
- 5Kuhn, J. H. Filoviruses: A Compendium of 40 Years of Epidemiological, Clinical, and Laboratory Studies; Calisher, C. H., Ed.; Springer: Wien, Austria, 2008.There is no corresponding record for this reference.
- 6Rougeron, V.; Feldmann, H.; Grard, G.; Becker, S.; Leroy, E. M. Ebola and Marburg Haemorrhagic Fever. J. Clin. Virol. 2015, 64, 111– 119, DOI: 10.1016/j.jcv.2015.01.0146Ebola and Marburg haemorrhagic feverRougeron V; Feldmann H; Grard G; Becker S; Leroy E MJournal of clinical virology : the official publication of the Pan American Society for Clinical Virology (2015), 64 (), 111-9 ISSN:.Ebolaviruses and Marburgviruses (family Filoviridae) are among the most virulent pathogens for humans and great apes causing severe haemorrhagic fever and death within a matter of days. This group of viruses is characterized by a linear, non-segmented, single-stranded RNA genome of negative polarity. The overall burden of filovirus infections is minimal and negligible compared to the devastation caused by malnutrition and other infectious diseases prevalent in Africa such as malaria, dengue or tuberculosis. In this paper, we review the knowledge gained on the eco/epidemiology, the pathogenesis and the disease control measures for Marburg and Ebola viruses developed over the last 15 years. The overall progress is promising given the little attention that these pathogen have achieved in the past; however, more is to come over the next decade given the more recent interest in these pathogens as potential public and animal health concerns. Licensing of therapeutic and prophylactic options may be achievable over the next 5-10 years.
- 7Qiu, X.; Wong, G.; Audet, J.; Bello, A.; Fernando, L.; Alimonti, J. B.; Fausther-Bovendo, H.; Wei, H.; Aviles, J.; Hiatt, E.; Johnson, A.; Morton, J.; Swope, K.; Bohorov, O.; Bohorova, N.; Goodman, C.; Kim, D.; Pauly, M. H.; Velasco, J.; Pettitt, J.; Olinger, G. G.; Whaley, K.; Xu, B.; Strong, J. E.; Zeitlin, L.; Kobinger, G. P. Reversion af Advanced Ebola Virus Disease in Nonhuman Primates with ZMapp. Nature 2014, 514, 47– 53, DOI: 10.1038/nature137777Reversion of advanced Ebola virus disease in nonhuman primates with ZMappQiu, Xiangguo; Wong, Gary; Audet, Jonathan; Bello, Alexander; Fernando, Lisa; Alimonti, Judie B.; Fausther-Bovendo, Hugues; Wei, Haiyan; Aviles, Jenna; Hiatt, Ernie; Johnson, Ashley; Morton, Josh; Swope, Kelsi; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Pauly, Michael H.; Velasco, Jesus; Pettitt, James; Olinger, Gene G.; Whaley, Kevin; Xu, Bianli; Strong, James E.; Zeitlin, Larry; Kobinger, Gary P.Nature (London, United Kingdom) (2014), 514 (7520), 47-53CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. A combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viremia and abnormalities in blood count and blood chem. were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal hemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clin. use.
- 8Thi, E. P.; Mire, C. E.; Lee, A. C. H.; Geisbert, J. B.; Zhou, J. Z.; Agans, K. N.; Snead, N. M.; Deer, D. J.; Barnard, T. R.; Fenton, K. A.; MacLachlan, I.; Geisbert, T. W. Lipid Nanoparticles siRNA Treatment of Ebola-Virus-Makona-Infected Nonhuman Primates. Nature 2015, 521, 362– 365, DOI: 10.1038/nature144428Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primatesThi, Emily P.; Mire, Chad E.; Lee, Amy C. H.; Geisbert, Joan B.; Zhou, Joy Z.; Agans, Krystle N.; Snead, Nicholas M.; Deer, Daniel J.; Barnard, Trisha R.; Fenton, Karla A.; MacLachlan, Ian; Geisbert, Thomas W.Nature (London, United Kingdom) (2015), 521 (7552), 362-365CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viremic and clin. ill. Although all infected animals showed evidence of advanced disease including abnormal haematol., blood chem. and coagulopathy, siRNA-treated animals had milder clin. features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.
- 9Tekmira Pharmaceuticals Corp. Tekmira Provides Update on TKM-Ebola-Guinea. http://www.sec.gov/Archives/edgar/data/1447028/000117184315003522/newsrelease.htm, 2015 (accessed Jul. 22, 2016).There is no corresponding record for this reference.
- 10Iversen, P. L.; Warren, T. K.; Wells, J. B.; Garza, N. L.; Mourich, D. V.; Welch, L. S.; Panchal, R. G.; Bavari, S. Discovery and Early Development of AVI-7537 and AVI-7288 For the Treatment of Ebola Virus and Marburg Virus Infections. Viruses 2012, 4, 2806– 2830, DOI: 10.3390/v411280610Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infectionsIversen, Patrick L.; Warren, Travis K.; Wells, Jay B.; Garza, Nicole L.; Mourich, Dan V.; Welch, Lisa S.; Panchal, Rekha G.; Bavari, SinaViruses (2012), 4 (), 2806-2830CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus resp. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chem. modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus contg. a limited no. of pos. charged linkages in the PMO structure. The initial lead compds. were combinations of two agents targeting sep. genes. In the final anal., a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus and are now progressing into late stage clin. development as the optimal therapeutic candidates.
- 11Oestereich, L.; Lüdtke, A.; Wurr, S.; Rieger, T.; Muñoz-Fontela, C.; Günther, S. Successful Treatment of Advanced Ebola Virus Infection With T-705 (favipiravir) in a Small Animal Model. Antiviral Res. 2014, 105, 17– 21, DOI: 10.1016/j.antiviral.2014.02.01411Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal modelOestereich, Lisa; Ludtke, Anja; Wurr, Stephanie; Rieger, Toni; Munoz-Fontela, Cesar; Gunther, StephanAntiviral Research (2014), 105 (), 17-21CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Outbreaks of Ebola hemorrhagic fever in sub-Saharan Africa are assocd. with case fatality rates of up to 90%. Currently, neither a vaccine nor an effective antiviral treatment is available for use in humans. Here, we evaluated the efficacy of the pyrazinecarboxamide deriv. T-705 (favipiravir) against Zaire Ebola virus (EBOV) in vitro and in vivo. T-705 suppressed replication of Zaire EBOV in cell culture by 4 log units with an IC90 of 110 μM. Mice lacking the type I interferon receptor (IFNAR-/-) were used as in vivo model for Zaire EBOV-induced disease. Initiation of T-705 administration at day 6 post infection induced rapid virus clearance, reduced biochem. parameters of disease severity, and prevented a lethal outcome in 100% of the animals. The findings suggest that T-705 is a candidate for treatment of Ebola hemorrhagic fever.
- 12Smither, S. J.; Eastaugh, L. S.; Steward, J. A.; Nelson, M.; Lenk, R. P.; Lever, M. S. Post-exposure Efficacy of Oral T-705 (Favipiravir) Against Inhalational Ebola Virus Infection in a Mouse Model. Antiviral Res. 2014, 104, 153– 155, DOI: 10.1016/j.antiviral.2014.01.01212Post-exposure efficacy of Oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse modelSmither, Sophie J.; Eastaugh, Lin S.; Steward, Jackie A.; Nelson, Michelle; Lenk, Robert P.; Lever, Mark S.Antiviral Research (2014), 104 (), 153-155CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Filoviruses cause disease with high case fatality rates and are considered biol. threat agents. Licensed post-exposure therapies that can be administered by the oral route are desired for safe and rapid distribution and uptake in the event of exposure or outbreaks. Favipiravir or T-705 has broad antiviral activity and has already undergone phase II and is undergoing phase III clin. trials for influenza. Here we report the first use of T-705 against Ebola virus. T-705 gave 100% protection against aerosol Ebola virus E718 infection; protection was shown in immune-deficient mice after 14 days of twice-daily dosing. T-705 was also shown to inhibit Ebola virus infection in cell culture. T-705 is likely to be licensed for use against influenza in the near future and could also be used with a new indication for filovirus infection.
- 13Sissoko, D.; Folkesson, E.; Abdoul, M.; Beavogui, A. H.; Gunther, S.; Shepherd, S.; Danel, C.; Mentre, F.; Anglaret, X.; Malvy, D. Favipiravir in Patients With Ebola Virus Disease: Early Results of the JIKI Trial in Guinea. Conference on Retroviruses and Opportunistic Infections, Seattle, WA, USA, Feb. 23–26, 2015, Abstract 103-ALB; CROI Foundation/IAS-USA: San Francisco, CA, USA, 2015.There is no corresponding record for this reference.
- 14McMullan, L. K.; Flint, M.; Dyall, J.; Albariño, C.; Olinger, G. G.; Foster, S.; Sethna, P.; Hensley, L. E.; Nichol, S. T.; Lanier, E. R.; Spiropoulou, C. F. The Lipid Moiety of Brincidofovir is Required For In Vitro Antiviral Activity Against Ebola Virus. Antiviral Res. 2016, 125, 71– 78, DOI: 10.1016/j.antiviral.2015.10.01014The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virusMcMullan, Laura K.; Flint, Mike; Dyall, Julie; Albarino, Cesar; Olinger, Gene G.; Foster, Scott; Sethna, Phiroze; Hensley, Lisa E.; Nichol, Stuart T.; Lanier, E. Randall; Spiropoulou, Christina F.Antiviral Research (2016), 125 (), 71-78CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was obsd. for several HDP-nucleotide conjugates.
- 15Warren, T. K.; Wells, J.; Panchal, R. G.; Stuthman, K. S.; Garza, N. L.; Van Tongeren, S. A.; Dong, L.; Retterer, C. J.; Eaton, B. P.; Pegoraro, G.; Honnold, S.; Bantia, S.; Kotian, P.; Chen, X.; Taubenheim, B. R.; Welch, L. S.; Minning, D. M.; Babu, Y. S.; Sheridan, W. P.; Bavari, S. Protection Against Filovirus Disease by a Novel Broad-Spectrum Nucleoside Analogue BCX4430. Nature 2014, 508, 402– 405, DOI: 10.1038/nature1302715Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430Warren, Travis K.; Wells, Jay; Panchal, Rekha G.; Stuthman, Kelly S.; Garza, Nicole L.; Van Tongeren, Sean A.; Dong, Lian; Retterer, Cary J.; Eaton, Brett P.; Pegoraro, Gianluca; Honnold, Shelley; Bantia, Shanta; Kotian, Pravin; Chen, Xilin; Taubenheim, Brian R.; Welch, Lisa S.; Minning, Dena M.; Babu, Yarlagadda S.; Sheridan, William P.; Bavari, SinaNature (London, United Kingdom) (2014), 508 (7496), 402-405CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analog, inhibits infection of distinct filoviruses in human cells. Biochem., reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure i.m. administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 h after infection. In addn., BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small mol. We provide addnl. pharmacol. characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.
- 16Henao-Restrepo, A. M.; Longini, I. M.; Egger, M.; Dean, N. E.; Edmunds, W. J.; Camacho, A.; Carroll, M. W.; Doumbia, M.; Draguez, B.; Duraffour, S.; Enwere, G.; Grais, R.; Gunther, S.; Hossmann, S.; Kondé, M. K.; Kone, S.; Kuisma, E.; Levine, M. M.; Mandal, S.; Norheim, G.; Riveros, X.; Soumah, A.; Trelle, S.; Vicari, A. S.; Watson, C. H.; Kéïta, S.; Kieny, M. P.; Røttingen, J.-A. Efficacy and Effectiveness of an rVSV-Vectored Vaccine Expressing Ebola, Surface Glycoprotein: Interim Results From the Guinea Ring Vaccination Cluster-Randomised Trial. Lancet 2015, 386, 857– 866, DOI: 10.1016/S0140-6736(15)61117-516Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trialHenao-Restrepo, Ana Maria; Longini, Ira M.; Egger, Matthias; Dean, Natalie E.; Edmunds, W. John; Camacho, Anton; Carroll, Miles W.; Doumbia, Moussa; Draguez, Bertrand; Duraffour, Sophie; Enwere, Godwin; Grais, Rebecca; Gunther, Stephan; Hossmann, Stefanie; Konde, Mandy Kader; Kone, Souleymane; Kuisma, Eeva; Levine, Myron M.; Mandal, Sema; Norheim, Gunnstein; Riveros, Ximena; Soumah, Aboubacar; Trelle, Sven; Vicari, Andrea S.; Watson, Conall H.; Keita, Sakoba; Kieny, Marie Paule; Roettingen, John-ArneLancet (2015), 386 (9996), 857-866CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)A recombinant, replication-competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Zaire Ebolavirus (rVSV-ZEBOV) is a promising Ebola vaccine candidate. We report the results of an interim anal. of a trial of rVSV-ZEBOV in Guinea, west Africa. For this open-label, cluster-randomised ring vaccination trial, suspected cases of Ebola virus disease (Guinea, west Africa) were independently ascertained by Ebola response teams as part of a national surveillance system. After lab. confirmation of a new case, clusters of all contacts and contacts of contacts were defined and randomly allocated 1:1 to immediate vaccination or delayed (21 days later) vaccination with rVSV-ZEBOV (one dose of 2 × 107 plaque-forming units, administered i.m. in the deltoid muscle). Adults (age ≥18 years) who were not pregnant or breastfeeding were eligible for vaccination. Block randomisation was used, with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 vs >20 individuals). The study is open label and masking of participants and field teams to the time of vaccination is not possible, but Ebola response teams and lab. workers were unaware of allocation to immediate or delayed vaccination. Taking into account the incubation period of the virus of about 10 days, the prespecified primary outcome was lab.-confirmed Ebola virus disease with onset of symptoms at least 10 days after randomisation. The primary anal. was per protocol and compared the incidence of Ebola virus disease in eligible and vaccinated individuals in immediate vaccination clusters with the incidence in eligible individuals in delayed vaccination clusters. This trial is registered with the Pan African Clin. Trials Registry, no. PACTR201503001057193. Between Apr. 1, 2015, and July 20, 2015, 90 clusters, with a total population of 7651 people were included in the planned interim anal. 48 of these clusters (4123 people) were randomly assigned to immediate vaccination with rVSV-ZEBOV, and 42 clusters (3528 people) were randomly assigned to delayed vaccination with rVSV-ZEBOV. In the immediate vaccination group, there were no cases of Ebola virus disease with symptom onset at least 10 days after randomisation, whereas in the delayed vaccination group there were 16 cases of Ebola virus disease from seven clusters, showing a vaccine efficacy of 100% (95% CI 74·7-100·0; p=0·0036). No new cases of Ebola virus disease were diagnosed in vaccinees from the immediate or delayed groups from 6 days post-vaccination. At the cluster level, with the inclusion of all eligible adults, vaccine effectiveness was 75·1% (95% CI -7·1 to 94·2; p=0·1791), and 76·3% (95% CI -15·5 to 95·1; p=0·3351) with the inclusion of everyone (eligible or not eligible for vaccination). 43 serious adverse events were reported; one serious adverse event was judged to be causally related to vaccination (a febrile episode in a vaccinated participant, which resolved without sequelae). Assessment of serious adverse events is ongoing. The results of this interim anal. indicate that rVSV-ZEBOV might be highly efficacious and safe in preventing Ebola virus disease, and is most likely effective at the population level when delivered during an Ebola virus disease outbreak via a ring vaccination strategy.
- 17Warren, T. K.; Jordan, R.; Lo, M. K.; Ray, A. S.; Mackman, R. L.; Soloveva, V.; Siegel, D.; Perron, M.; Bannister, R.; Hui, H. C.; Larson, N.; Strickley, R.; Wells, J.; Stuthman, K. S.; Van Tongeren, S. A.; Garza, N. L.; Donnelly, G.; Shurtleff, A. C.; Retterer, C. J.; Gharaibeh, D.; Zamani, R.; Kenny, T.; Eaton, B. P.; Grimes, E.; Welch, L. S.; Gomba, L.; Wilhelmsen, C. L.; Nichols, D. K.; Nuss, J. E.; Nagle, E. R.; Kugelman, J. R.; Palacios, G.; Doerffler, E.; Neville, S.; Carra, E.; Clarke, M. O.; Zhang, L.; Lew, W.; Ross, B.; Wang, Q.; Chun, K.; Wolfe, L.; Babusis, D.; Park, Y.; Stray, K. M.; Trancheva, I.; Feng, J. Y.; Barauskas, O.; Xu, Y.; Wong, P.; Braun, M. R.; Flint, M.; McMullan, L. K.; Chen, S. S.; Fearns, R.; Swaminathan, S.; Mayers, D. L.; Spiropoulou, C. F.; Lee, W. A.; Nichol, S. T.; Cihlar, T.; Bavari, S. Therapeutic Efficacy of The Small Molecule GS-5734 Against Ebola Virus in Rhesus Monkeys. Nature 2016, 531, 381– 385, DOI: 10.1038/nature1718017Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeysWarren, Travis K.; Jordan, Robert; Lo, Michael K.; Ray, Adrian S.; Mackman, Richard L.; Soloveva, Veronica; Siegel, Dustin; Perron, Michel; Bannister, Roy; Hui, Hon C.; Larson, Nate; Strickley, Robert; Wells, Jay; Stuthman, Kelly S.; Van Tongeren, Sean A.; Garza, Nicole L.; Donnelly, Ginger; Shurtleff, Amy C.; Retterer, Cary J.; Gharaibeh, Dima; Zamani, Rouzbeh; Kenny, Tara; Eaton, Brett P.; Grimes, Elizabeth; Welch, Lisa S.; Gomba, Laura; Wilhelmsen, Catherine L.; Nichols, Donald K.; Nuss, Jonathan E.; Nagle, Elyse R.; Kugelman, Jeffrey R.; Palacios, Gustavo; Doerffler, Edward; Neville, Sean; Carra, Ernest; Clarke, Michael O.; Zhang, Lijun; Lew, Willard; Ross, Bruce; Wang, Queenie; Chun, Kwon; Wolfe, Lydia; Babusis, Darius; Park, Yeojin; Stray, Kirsten M.; Trancheva, Iva; Feng, Joy Y.; Barauskas, Ona; Xu, Yili; Wong, Pamela; Braun, Molly R.; Flint, Mike; McMullan, Laura K.; Chen, Shan-Shan; Fearns, Rachel; Swaminathan, Swami; Mayers, Douglas L.; Spiropoulou, Christina F.; Lee, William A.; Nichol, Stuart T.; Cihlar, Tomas; Bavari, SinaNature (London, United Kingdom) (2016), 531 (7594), 381-385CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The most recent Ebola virus outbreak in West Africa, which was unprecedented in the no. of cases and fatalities, geog. distribution, and no. of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clin. efficacy. Here we report the discovery of a novel small mol. GS-5734, a monophosphoramidate prodrug of an adenosine analog, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacol. active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. I.v. administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily i.v. administration of 10 mg kg-1 GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clin. disease signs and pathophysiol. markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-mol. antiviral compd. against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufg., and clin. studies investigating the drug safety and pharmacokinetics are ongoing.
- 18Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy Phosphoramidate Triesters: A Technology For Delivering Monophosphorylated Nucleosides and Sugars Into Cells. ChemMedChem 2009, 4, 1779– 1791, DOI: 10.1002/cmdc.20090028918Aryloxy Phosphoramidate Triesters: a Technology for Delivering Monophosphorylated Nucleosides and Sugars into CellsMehellou, Youcef; Balzarini, Jan; McGuigan, ChristopherChemMedChem (2009), 4 (11), 1779-1791CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Prodrug technologies aimed at delivering nucleoside monophosphates into cells (protides) have proved to be effective in improving the therapeutic potential of antiviral and anticancer nucleosides. In these cases, the nucleoside monophosphates are delivered into the cell, where they may then be further converted (phosphorylated) to their active species. Herein, we describe one of these technologies developed in our labs., known as the phosphoramidate protide method. In this approach, the charges of the phosphate group are fully masked to provide efficient passive cell-membrane penetration. Upon entering the cell, the masking groups are enzymically cleaved to release the phosphorylated biomol. The application of this technol. to various therapeutic nucleosides has resulted in improved antiviral and anticancer activities, and in some cases it has transformed inactive nucleosides to active ones. Addnl., the phosphoramidate technol. has also been applied to numerous antiviral nucleoside phosphonates, and has resulted in at least three phosphoramidate-based nucleotides progressing to clin. investigations. Furthermore, the phosphoramidate technol. has been recently applied to sugars (mainly glucosamine) in order to improve their therapeutic potential. The development of the phosphoramidate technol., mechanism of action and the application of the technol. to various monophosphorylated nucleosides and sugars will be reviewed.
- 19Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.; Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang, H.-R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a β-D-2′-Deoxy-2′-α-Fluoro-2′-β-C-Methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C Virus. J. Med. Chem. 2010, 53, 7202– 7218, DOI: 10.1021/jm100863x19Discovery of a β-D-2'-Deoxy-2'-α-fluoro-2'-β-C-methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C VirusSofia, Michael J.; Bao, Donghui; Chang, Wonsuk; Du, Jinfa; Nagarathnam, Dhanapalan; Rachakonda, Suguna; Reddy, P. Ganapati; Ross, Bruce S.; Wang, Peiyuan; Zhang, Hai-Ren; Bansal, Shalini; Espiritu, Christine; Keilman, Meg; Lam, Angela M.; Steuer, Holly M. Micolochick; Niu, Congrong; Otto, Michael J.; Furman, Phillip A.Journal of Medicinal Chemistry (2010), 53 (19), 7202-7218CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. β-D-2'-Deoxy-2'-α-fluoro-2'-β-C-Me nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5'-phosphate deriv. of the β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methyluridine nucleoside were prepd. and showed significant potency in the HCV subgenomic replicon assay (<1 μM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixt. 14 was crystd., and an X-ray structure was detd. establishing the phosphoramidate stereochem. as Sp, thus correlating for the first time the stereochem. of a phosphoramidate prodrug with biol. activity. 51 (PSI-7977) was selected as a clin. development candidate.
- 20Lee, A. W.; He, G.-X.; Eisenberg, E.; Cihlar, T.; Swaminathan, S.; Mulato, A.; Cundy, K. C. Selective Intracellular Activation of a Novel Prodrug of the Human Immunodeficiency Virus Reverse Transcriptase Inhibitor Tenofovir Leads to Preferential Distribution and Accumulation in Lymphatic Tissue. Antimicrob. Agents Chemother. 2005, 49, 1898– 1906, DOI: 10.1128/AAC.49.5.1898-1906.200520Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissueLee, William A.; He, Gong-Xin; Eisenberg, Eugene; Cihlar, Tomas; Swaminathan, Swami; Mulato, Andrew; Cundy, Kenneth C.Antimicrobial Agents and Chemotherapy (2005), 49 (5), 1898-1906CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)An isopropylalaninyl monoamidate Ph monoester prodrug of tenofovir (GS 7340) was prepd., and its in vitro antiviral activity, metab., and pharmacokinetics in dogs were detd. The 50% effective concn. (EC50) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 μM compared to an EC50 of 5 μM for the parent drug, tenofovir. The (L)-alaninyl analog (GS 7340) was >1000-fold more active than the (D)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at 37° and a half-life of 28.3 min in an MT-2 cell ext. at 37°. The antiviral activity (>10× the EC50) and the metabolic stability in MT-2 cell exts. (>35×) and plasma (>2.5×) were also sensitive to the stereochem. at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs, the plasma bioavailability of tenofovir compared to an i.v. dose of tenofovir was 17%. The total intracellular concn. of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was 63 μg-eq/mL compared to 0.2 μg-eq/mL in plasma. A radiolabeled distribution study with dogs resulted in an increased distribution of tenofovir to tissues of lymphatic origin compared to the com. available prodrug tenofovir DF (Viread).
- 21Murakami, E.; Niu, C.; Bao, H.; Steuer, H. M. M.; Whitaker, T.; Nachman, T.; Sofia, M. A.; Wang, P.; Otto, M. J.; Furman, P. A. The Mechanism of Action of β-D-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine Involves a Second Metabolic Pathway Leading to β-D-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine 5′-Triphosphate, A Potent Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase. Antimicrob. Agents Chemother. 2008, 52, 458– 464, DOI: 10.1128/AAC.01184-0721The mechanism of action of β-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine involves a second metabolic pathway leading to β-D-2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-triphosphate, a potent inhibitor of the hepatitis C virus RNA-dependent RNA polymeraseMurakami, Eisuke; Niu, Congrong; Bao, Haiying; Micolochick Steuer, Holly M.; Whitaker, Tony; Nachman, Tammy; Sofia, Michael A.; Wang, Peiyuan; Otto, Michael J.; Furman, Phillip A.Antimicrobial Agents and Chemotherapy (2008), 52 (2), 458-464CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)β-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metab. of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, β-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, resp. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.
- 22Cho, A.; Saunders, O. L.; Butler, T.; Zhang, L.; Xu, J.; Vela, J. E.; Feng, J. Y.; Ray, A. S.; Kim, C. U. Synthesis and Antiviral Activity of a Series of 1′-Substituted 4-Aza-7,9-Dideazaadenosine C-Nucleosides. Bioorg. Med. Chem. Lett. 2012, 22, 2705– 2707, DOI: 10.1016/j.bmcl.2012.02.10522Synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine C-nucleosidesCho, Aesop; Saunders, Oliver L.; Butler, Thomas; Zhang, Lijun; Xu, Jie; Vela, Jennifer E.; Feng, Joy Y.; Ray, Adrian S.; Kim, Choung U.Bioorganic & Medicinal Chemistry Letters (2012), 22 (8), 2705-2707CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)A series of 1'-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside, e.g. I, were prepd. and evaluated for the potential as antiviral agents. These compds. showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R = CN was attributed to inhibition of HCV NS5B polymerase and intracellular concn. of the corresponding nucleoside triphosphate.
- 23Mackman, R. L.; Parrish, J. P.; Ray, A. S.; Theodore, D. A. Methods and Compounds for Treating Paramyxoviridae Virus Infections. U.S. Patent 2011045102 July, 22, 2011.There is no corresponding record for this reference.
- 24Patil, S. A.; Otter, P. B.; Klein, R. S. 4-Aza-7,9-Dideazaadenosine, A New Cytotoxic Synthetic C-Nucleoside Analogue of Adenosine. Tetrahedron Lett. 1994, 35, 5339– 5342, DOI: 10.1016/S0040-4039(00)73494-0244-Aza-7,9-dideazaadenosine, a new cytotoxic synthetic C-nucleoside analog of adenosinePatil, Shirish A.; Otter, Brian A.; Klein, Robert S.Tetrahedron Letters (1994), 35 (30), 5339-42CODEN: TELEAY; ISSN:0040-4039.The first synthesis of I, the pyrrolo[2,1-f]triazine C-nucleoside congener of adenosine is described. The key intermediate ribofuranosyl pyrrole is obtained by the direct C-ribosylation of pyrrolemagnesium bromide followed by an acid-catalyzed dehydration, Vilsmeier formylation, and N-amination. In vitro growth inhibitory activities of I against leukemic cell lines (0.8-15 nM) are comparable to those of 9-deazaadenosine.
- 25Lou, Z.; Chen, G.; Xie, Y. Cyanoribofuranoside Compound and a Preparation Method Thereof. Chinese Patent CN 1137132 C Feb., 4, 2004.There is no corresponding record for this reference.
- 26Yoshimura, Y.; Kano, F.; Miyazaki, S.; Ashida, N.; Sakata, S.; Haraguchi, K.; Itoh, Y.; Tanaka, H.; Miyasaka, T. Synthesis and Biological Evaluation of 1′-C-Cyano-Pyrimidine Nucleosides. Nucleosides, Nucleotides Nucleic Acids 1996, 15, 305– 324, DOI: 10.1080/07328319608002386There is no corresponding record for this reference.
- 27Kirschberg, T. A.; Mish, M.; Squires, N. H.; Zonte, S.; Aktoudianakis, E.; Metobo, S.; Butler, T.; Ju, X.; Cho, A.; Ray, A. S.; Kim, C. U. Synthesis of 1′-C-Cyano Pyrimidine Nucleosides and Characterization as HCV Polymerase Inhibitors. Nucleosides, Nucleotides Nucleic Acids 2015, 34, 763– 785, DOI: 10.1080/15257770.2015.107555027Synthesis of 1'-C-Cyano Pyrimidine Nucleosides and Characterization as HCV Polymerase InhibitorsKirschberg, Thorsten A.; Mish, Michael; Squires, Neil H.; Zonte, Sebastian; Aktoudianakis, Evangelos; Metobo, Sammy; Butler, Thomas; Ju, Xie; Cho, Aesop; Ray, Adrian S.; Kim, Choung U.Nucleosides, Nucleotides & Nucleic Acids (2015), 34 (11), 763-785CODEN: NNNAFY; ISSN:1525-7770. (Taylor & Francis Ltd.)Ribose modified 1'-C-cyano pyrimidine nucleosides were synthesized. A silver triflate mediated Vorbruggen reaction was used to generate the nucleoside scaffold and follow-up chem. provided specific ribose modified analogs. Nucleosides and phosphoramidate prodrugs were tested for their anti-HCV activity.
- 28Cho, A.; Zhang, L.; Xu, J.; Lee, R.; Butler, T.; Metobo, S.; Aktoudianakis, V.; Lew, W.; Ye, H.; Clarke, M.; Doerffler, E.; Byun, D.; Wang, T.; Babusis, D.; Carey, A. C.; German, P.; Sauer, D.; Zhong, W.; Rossi, S.; Fenaux, M.; McHutchison, J. G.; Perry, J.; Feng, J.; Ray, A. S.; Kim, C. U. Discovery of the First C-nucleoside HCV Polymerase Inhibitor (GS-6620) with Demonstrated Antiviral Response in HCV Infected Patients. J. Med. Chem. 2014, 57, 1812– 1825, DOI: 10.1021/jm400201a28Discovery of the First C-Nucleoside HCV Polymerase Inhibitor (GS-6620) with Demonstrated Antiviral Response in HCV Infected PatientsCho, Aesop; Zhang, Lijun; Xu, Jie; Lee, Rick; Butler, Thomas; Metobo, Sammy; Aktoudianakis, Vangelis; Lew, Willard; Ye, Hong; Clarke, Michael; Doerffler, Edward; Byun, Daniel; Wang, Ting; Babusis, Darius; Carey, Anne C.; German, Polina; Sauer, Dorothea; Zhong, Weidong; Rossi, Stephen; Fenaux, Martijn; McHutchison, John G.; Perry, Jason; Feng, Joy; Ray, Adrian S.; Kim, Choung U.Journal of Medicinal Chemistry (2014), 57 (5), 1812-1825CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clin. development thus far have all been N-nucleoside derivs. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-Me 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compds. showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clin. development candidate GS-6620 (I). In a phase I clin. study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.
- 29(a) Smith, J. T.; Elkin, J. T.; Reichert, W. M. Directed Cell Migration on Fibronectin Gradients: Effect of Gradient Slope. Exp. Cell Res. 2006, 312, 2424– 2432, DOI: 10.1016/j.yexcr.2006.04.00529aDirected cell migration on fibronectin gradients: Effect of gradient slopeSmith, Jason T.; Elkin, James T.; Reichert, W. MontyExperimental Cell Research (2006), 312 (13), 2424-2432CODEN: ECREAL; ISSN:0014-4827. (Elsevier)The migration of human microvascular endothelial cells (hMEC) was measured on a range of fibronectin gradient slopes. hMEC drift speed increased with increasing gradient slope with no concurrent change in cellular persistence time or random cell speed. The frequency of discrete cellular motion in the gradient direction increased with gradient slope. Morphol. polarization of cells on the gradients is also characterized and correlated with cellular drift speed. These expts. present the first demonstration of cellular response to changing haptotactic gradient slope using an in vitro system for the quant. study of cell migration.(b) Zhao, L.; Kroenke, C. D.; Song, J.; Piwnica-Worms, D.; Ackerman, J. J. H.; Neil, J. J. Intracellular Water Specific MR of Microbead-Adherent Cells: The HeLa Cell Intracellular Water Exchange Lifetime. NMR Biomed. 2008, 21, 159– 164, DOI: 10.1002/nbm.117329bIntracellular water-specific MR of microbead-adherent cells: the HeLa cell intracellular water exchange lifetimeZhao, L.; Kroenke, C. D.; Song, J.; Piwnica-Worms, D.; Ackerman, J. J. H.; Neil, J. J.NMR in Biomedicine (2008), 21 (2), 159-164CODEN: NMRBEF; ISSN:0952-3480. (John Wiley & Sons Ltd.)Quant. characterization of the intracellular water 1H MR signal from cultured cells will provide crit. biophys. insight into the MR signal from tissues in vivo. Microbeads provide a robust immobilization substrate for the m any mammalian cell lines that adhere to surfaces and also provide sufficient cell d. for observation of the intracellular water MR signal. However, selective observation of the intracellular water MR signal from perfused, microbead-adherent mammalian cells requires highly effective suppression of the extracellular water MR signal. We describe how high-velocity perfusion of microbead-adherent cells results in short apparent 1H MR longitudinal and transverse relaxation times for the extracellular water in a thin slice selected orthogonal to the direction of flow. When combined with a spin-echo pulse sequence, this phenomenon provides highly effective suppression of the extracellular water MR signal. This new method is exploited here to quantify the kinetics of water exchange from the intracellular to extracellular spaces of HeLa cells. The time const. describing water exchange from intracellular to extracellular spaces, also known as the exchange lifetime for intracellular water, is 119 ± 14 ms.
- 30Clarke, M. O.; Mackman, R.; Byun, D.; Hui, H.; Barauskas, O.; Birkus, G.; Chun, B.-K.; Doerffler, E.; Feng, J.; Karki, K.; Lee, G.; Perron, M.; Siegel, D.; Swaminathan, S.; Lee, W. Discovery of β-D-2′-α-Fluoro-4′-α-Cyano-5-Aza-7,9-Dideaza Adenosine as a Potent Nucleoside Inhibitor of Respiratory Syncytial Virus With Excellent Selectivity Over Mitochondrial RNA and DNA Polymerases. Bioorg. Med. Chem. Lett. 2015, 25, 2484– 2487, DOI: 10.1016/j.bmcl.2015.04.07330Discovery of β-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerasesClarke, Michael O.; Mackman, Richard; Byun, Daniel; Hui, Hon; Barauskas, Ona; Birkus, Gabriel; Chun, Byoung-Kwon; Doerffler, Edward; Feng, Joy; Karki, Kapil; Lee, Gary; Perron, Michel; Siegel, Dustin; Swaminathan, Swami; Lee, WilliamBioorganic & Medicinal Chemistry Letters (2015), 25 (12), 2484-2487CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Novel 4'-substituted β-D-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compds. demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of I as a promising nucleoside lead for RSV.
- 31Feng, J.; Xu, Y.; Barauskas, O.; Perry, J. K.; Ahmadyar, S.; Stepan, G.; Yu, H.; Babusis, D.; Park, Y.; McCutcheon, K.; Perron, M.; Schultz, B. E.; Sakowicz, R.; Ray, A. S. Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus. Antimicrob. Agents Chemother. 2016, 60, 806– 817, DOI: 10.1128/AAC.01922-1531Role of mitochondrial RNA polymerase in the toxicity of nucleotide inhibitors of hepatitis C virusFeng, Joy Y.; Xu, Yili; Barauskas, Ona; Perry, Jason K.; Ahmadyar, Shekeba; Stepan, George; Yu, Helen; Babusis, Darius; Park, Yeojin; McCutcheon, Krista; Perron, Michel; Schultz, Brian E.; Sakowicz, Roman; Ray, Adrian S.Antimicrobial Agents and Chemotherapy (2016), 60 (2), 806-817CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)Toxicity has emerged during the clin. development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clin. relevant HCV NI were characterized in biochem. and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4'-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2'-C-Me guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concns., followed by general cytotoxicity. In contrast, NI contg. multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2'-deoxynucleotide analogs, there is an assocn. between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clin. testing.
- 32(a) Müller, R.; Poch, O.; Delarue, M.; Bishop, D. H. L.; Bouloy, M. Rift Valley Fever Virus L Segment: Correction of the Sequence and Possible Functional Role of Newly Identified Regions Conserved in RNA-Dependent Polymerases. J. Gen. Virol. 1994, 75, 1345– 1352, DOI: 10.1099/0022-1317-75-6-134532aRift valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerasesMueller, R.; Poch, O.; Delarue, M.; Bishop, D. H. L.; Bouloy, M.Journal of General Virology (1994), 75 (6), 1345-52CODEN: JGVIAY; ISSN:0022-1317.The sequence of Rift Valley fever virus L segment that the authors published in a previous paper was erroneous in the 3'-terminal region of the antigenomic RNA mol. Here, the authors have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237.7 K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 neg.-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called polymerase module, the authors identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented neg.-stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA-dependent polymerases in the polymerase module, the authors have detd. the position of the conserved residues in these polymerases and discuss their possible functions in light of the variable structural information.(b) Huang, H.; Chopra, R.; Verdine, G. L.; Harrison, S. C. Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistance. Science 1998, 282, 1669– 1675, DOI: 10.1126/science.282.5394.166932bStructure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistanceHuang, Huifang; Chopra, Rajiv; Verdine, Gregory L.; Harrison, Stephen C.Science (Washington, D. C.) (1998), 282 (5394), 1669-1675CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A combinatorial disulfide crosslinking strategy was used to prep. a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase with a DNA template:primer and a deoxynucleoside triphosphate (dNTP), and the crystal structure of the complex was detd. at a resoln. of 3.2 angstroms. The presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the mol. basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chem. structure of the drug.(c) Appleby, T. C.; Perry, J. K.; Murakami, E.; Barauskas, O.; Feng, J.; Cho, A.; Fox, D.; Wetmore, D. R.; McGrath, M. E.; Ray, A. S.; Sofia, M. J.; Swaminathan, S.; Edwards, T. E. Structural Basis For RNA Replication by the Hepatitis C Virus Polymerase. Science 2015, 347, 771– 775, DOI: 10.1126/science.125921032cStructural basis for RNA replication by the hepatitis C virus polymeraseAppleby, Todd C.; Perry, Jason K.; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David, III; Wetmore, Diana R.; McGrath, Mary E.; Ray, Adrian S.; Sofia, Michael J.; Swaminathan, S.; Edwards, Thomas E.Science (Washington, DC, United States) (2015), 347 (6223), 771-775CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Nucleotide analog inhibitors have shown clin. success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by detg. crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our anal. revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clin. active metabolite formed by sofosbuvir, to elucidate key mol. interactions in the active site.
- 33
For active site models of other filovirus polymerases see the Supporting Information.
There is no corresponding record for this reference. - 34Feng, J. Y.; Cheng, G.; Perry, J.; Barauskas, O.; Xu, Y.; Fenaux, M.; Eng, S.; Tirunagari, N.; Peng, B.; Yu, M.; Tian, Y.; Lee, Y.-J.; Stepan, G.; Lagpacan, L. L.; Jin, D.; Hung, M.; Ku, K. S.; Han, B.; Kitrinos, K.; Perron, M.; Birkus, G.; Wong, K. A.; Zhong, W.; Kim, C. U.; Carey, A.; Cho, A.; Ray, A. S. Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C-Nucleoside Monophosphate Prodrug. Antimicrob. Agents Chemother. 2014, 58, 1930– 1942, DOI: 10.1128/AAC.02351-1334Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrugFeng, Joy Y.; Cheng, Guofeng; Perry, Jason; Barauskas, Ona; Xu, Yili; Fenaux, Martijn; Eng, Stacey; Tirunagari, Neeraj; Peng, Betty; Yu, Mei; Tian, Yang; Lee, Yu-Jen; Stepan, George; Lagpacan, Leanna L.; Jin, Debi; Hung, Magdeleine; Ku, Karin S.; Han, Bin; Kitrinos, Kathryn; Perron, Michel; Birkus, Gabriel; Wong, Kelly A.; Zhong, Weidong; Kim, Choung U.; Carey, Anne; Cho, Aesop; Ray, Adrian S.Antimicrobial Agents and Chemotherapy (2014), 58 (4), 1930-1942, 14 pp.CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacol. of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concn. [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, resp. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clin. trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.
- 35Butler, T.; Cho, A.; Kim, C. U.; Saunders, O. L.; Zhang, L. 1′-Substituted Carba-Nucleoside Analogs for Antiviral Treatment. U.S. Patent 2009041447 Apr., 22, 2009.There is no corresponding record for this reference.
- 36Butler, T.; Cho, A.; Graetz, B. R.; Kim, C. U.; Metobo, S. E.; Saunders, O. L.; Waltman, A. W.; Xu, J.; Zhang, L. Processes and Intermediates for the Preparation of 1′-Substituted Carba-Nucleoside Analogs. U.S. Patent20100459508 Sep., 20, 2010.There is no corresponding record for this reference.
- 37Metobo, S. E.; Xu, J.; Saunders, O. L.; Butler, T.; Aktoudianakis, E.; Cho, A.; Kim, C. U. Practical Synthesis of 1′-Substituted Tubercidin C-Nucleoside Analogs. Tetrahedron Lett. 2012, 53, 484– 486, DOI: 10.1016/j.tetlet.2011.11.05537Practical synthesis of 1'-substituted Tubercidin C-nucleoside analogsMetobo, Sammy E.; Xu, Jie; Saunders, Oliver L.; Butler, Thomas; Aktoudianakis, Evangelos; Cho, Aesop; Kim, Choung U.Tetrahedron Letters (2012), 53 (5), 484-486CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)Several 1'-substituted analogs of Tubercidin C-nucleosides were prepd. using a highly convergent synthesis. Good to high diastereoselectivity was achieved using a variety of nucleophiles targeting the 1'-position. The source for this stereoselectivity is herein proposed. It is thought to be attributed to a temp.-dependent chelation of the incoming nucleophile to either the 2'- or 3'-benzyloxy ether of the ribose core.
- 38Axt, S. D.; Badalov, P. R.; Brak, K.; Campagna, S.; Chtchemelinine, A.; Chun, B. K.; Clarke, M. O. H.; Doerffler, E.; Frick, M. M.; Gao, D.; Heumann, L. V.; Hoang, B.; Hui, H. C.; Jordan, R.; Lew, W.; Mackman, R. L.; Milburn, R. R.; Neville, S. T.; Parrish, J. P.; Ray, A. S.; Ross, B.; Rueden, E.; Scott, R. W.; Siegel, D.; Stevens, A. C.; Tadeus, C.; Vieira, T.; Waltman, A. W.; Wang, X.; Whitcomb, M. C.; Wolfe, L.; Yu, C.-Y. Methods For Treating Filoviridae Virus Infections. U.S. Patent 2015017934, Oct. 29, 2015.There is no corresponding record for this reference.
- 39Krasovskiy, A.; Knochel, P. A LiCl-Mediated Br/Mg Exchange Reaction For the Preparation of Functionalized Aryl- and Heteroarylmagnesium Compounds From Organic Bromides. Angew. Chem., Int. Ed. 2004, 43, 3333– 3336, DOI: 10.1002/anie.20045408439A LiCl-mediated Br/Mg exchange reaction for the preparation of functionalized aryl- and heteroarylmagnesium compounds from organic bromidesKrasovskiy, Arkady; Knochel, PaulAngewandte Chemie, International Edition (2004), 43 (25), 3333-3336CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A wide range of aryl and heteroaryl bromides, which are usually sluggish in exchange reactions, are readily converted into the corresponding Grignard reagents by means of a Br/Mg exchange reaction triggered by iPrMgCl·LiCl. These Grignard intermediates react with electrophiles in good yields. For example, 4-MeOC6H4MgCl·LiCl was prepd. from iPrMgCl·LiCl and 4-MeOC6H4Br and reacted with PhCHO giving 4-MeOC6H4CH(OH)Ph (70% yield).
- 40For a separate account describing the crystallization induced resolution of p-nitrophenolate 2-ethylbutyl-L-alaninate phosphoramidate see: Klasson, B.; Eneroth, A.; Nilson, M.; Pinho, P.; Samuelsson, B.; Sund, C. HCV Polymerase Inhibitors. European Patent IB2012056994 Dec. 5, 2012. The conditions in this patent were identified independently/concurrently with this report.There is no corresponding record for this reference.
- 41Bahar, F. G.; Ohura, K.; Ogihara, T.; Imai, T. Species Difference of Esterase Expression and Hydrolase Activity in Plasma. J. Pharm. Sci. 2012, 101, 3979– 3988, DOI: 10.1002/jps.2325841Species difference of esterase expression and hydrolase activity in plasmaBahar, Fatma Goksin; Ohura, Kayoko; Ogihara, Takuo; Imai, TerukoJournal of Pharmaceutical Sciences (2012), 101 (10), 3979-3988CODEN: JPMSAE; ISSN:0022-3549. (John Wiley & Sons, Inc.)Differences in esterase expression among human, rhesus monkey, cynomolgus monkey, dog, minipig, rabbit, rat, and mouse plasma were identified using native polyacrylamide gel electrophoresis. Paraoxonase (PON) and butyrylcholinesterase (BChE) were ubiquitous in all species, but were highly expressed in primates and dogs, whereas carboxylesterase (CES) was only abundant in rabbits, mice, and rats. Several unknown esterases were obsd. in minipig and mouse plasma. These differences in plasma esterases and their expression levels result in species differences with respect to hydrolase activity. These differences were characterized using several different substrates. In contrast to the high hydrolase activity found for p-nitrophenylacetate (PNPA), a substrate of several hydrolase enzymes, irinotecan, a carbamate compd., was resistant to all plasma esterases. Oseltamivir, temocapril, and propranolol (PL) derivs. were rapidly hydrolyzed in mouse and rat plasma by their highly active CES enzyme, but rabbit plasma CES hydrolyzed only the PL derivs. Interestingly, PL derivs. were highly hydrolyzed by monkey plasma BChE, whereas BChE from human, dog, and minipig plasma showed negligible activity. In conclusion, the esterase expression and hydrolyzing pattern of dog plasma were found to be closest to that of human plasma. These differences should be considered when selecting model animals for preclin. studies.
- 42Jacobs, M.; Rodger, A.; Bell, D. D.; Bhagani, S.; Cropley, I.; Filipe, A.; Gifford, R. J.; Hopkins, S.; Hughes, J.; Jabeen, F.; Johannessen, I.; Karageorgopoulos, D.; Lackenby, A.; Lester, R.; Liu, R. S. N.; MacConnachie, A.; Mahungu, T.; Martin, D.; Marshall, N.; Mepham, S.; Orton, R.; Palmarini, M.; Patel, M.; Perry, C.; Peters, S. E.; Porter, D.; Ritchie, D.; Ritchie, N. D.; Seaton, R. A.; Sreenu, V. B.; Templeton, K.; Warren, S.; Wilkie, G. S.; Zambon, M.; Gopal, R.; Thomson, E. C. Late Ebola Virus Relapse Causing Meningoencephalitis: A Case Report. Lancet 2016, 388, 498– 503, DOI: 10.1016/S0140-6736(16)30386-542Late Ebola virus relapse causing meningoencephalitis: a case reportJacobs Michael; Rodger Alison; Bell David J; MacConnachie Alisdair; Perry Colin; Peters S Erica; Porter Duncan; Ritchie David; Ritchie Neil D; Seaton R Andrew; Bhagani Sanjay; Cropley Ian; Hopkins Susan; Karageorgopoulos Drosos; Lester Rebecca; Mahungu Tabitha; Mepham Stephen; Warren Simon; Filipe Ana; Gifford Robert J; Hughes Joseph; Orton Richard; Palmarini Massimo; Sreenu Vattipally B; Wilkie Gavin S; Jabeen Farrah; Johannessen Ingolfur; Templeton Kate; Lackenby Angie; Zambon Maria; Liu Rebecca S N; Martin Daniel; Marshall Neal; Patel Monika; Gopal Robin; Thomson Emma CLancet (London, England) (2016), 388 (10043), 498-503 ISSN:.BACKGROUND: There are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13.2). METHODS: A 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach. FINDINGS: On admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23.7) than plasma (31.3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus. INTERPRETATION: Our report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern. FUNDING: Royal Free London NHS Foundation Trust.
- 43Schnirring, L. Youngest Ebola Survivor Leaves Guinea Hospital; Center For Infectious Disease Research and Policy, Minneapolis, MN, USA, Nov. 30, 2015; http://www.cidrap.umn.edu/news-perspective/2015/11/youngest-ebola-survivor-leaves-guinea-hospital (accessed Jul. 22, 2016).There is no corresponding record for this reference.
- 44National Institues of Health. PREVAIL Treatment Trial For Men With Persistent Ebola Viral RNA in Semen Opens in Liberia, Jul. 5, 2016; https://www.nih.gov/news-events/news-releases/prevail-treatment-trial-men-persistent-ebola-viral-rna-semen-opens-liberia (accessed, Jul., 22, 2016).There is no corresponding record for this reference.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01594.
Homology model of HIV (1RTD (32b)) X-ray structure used to generate the EBOV model for 4tp (PDB)
Homology model of HCV (4WTG (32c)) X-ray structure used to generate the EBOV model for 13tp (PDB)
Molecular formula strings (CSV)
Assay methods, molecular modeling with RSV, Marburg and Sudan viruses, compound synthesis, and single crystal X-ray structure information(PDF)
The Cambridge Crystallographic Data Center (CCDC) numbers for the X-ray structures of compound 22b and 4b are 1445315 and 1525480, respectively.
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.