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 Viruses

Article abstract of DOI:10.1021/acs.jmedchem.6b01594

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.

Full text of DOI:10.1021/acs.jmedchem.6b01594

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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 Viruses
Dustin Siegel, Hon C. Hui, Edward Doerffler, Michael O. Clarke, Kwon Chun, Lijun Zhang, Sean Neville,
Ernest Carra, Willard Lew, Bruce Saxby Ross, Queenie Wang, Lydia C. Wolfe, Robert Jordan, Veronica
Soloveva, John Edward Knox, Jason Perry, Michel Perron, Kirsten M. Stray, Ona Barauskas, Joy Y. Feng,
Yili Xu, Gary Lee, Arnold L. Rheingold, Adrian S. Ray, Roy Bannister, Robert Strickley, Swami Swaminathan,
William A. Lee, Sina Bavari, Tomas Cihlar, Michael K. Lo, Travis K. Warren, and Richard L. Mackman
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Jan 2017
Downloaded from http://pubs.acs.org on January 27, 2017
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Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1ꢀf][triazinꢀ4ꢀ
amino] Adenine CꢀNucleoside (GSꢀ5734) for the Treatment of Ebola and Emerging Viruses
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Dustin Siegel , Hon C. Hui , Edward Doerffler , Michael O. Clarke , Kwon Chun Lijun Zhang ,
,
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Sean Neville , Ernest Carra , Willard Lew , Bruce Ross , Queenie Wang , Lydia Wolfe , Robert
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Jordan , Veronica Soloveva , John Knox , Jason Perry , Michel Perron , Kirsten M. Stray , Ona
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Barauskas , Joy Y. Feng , Yili Xu , Gary Lee , Arnold L. Rheingold , Adrian S. Ray , Roy
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Bannister , Robert Strickley , Swami Swaminathan , William A. Lee , Sina Bavari , Tomas
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Cihlar , Michael K. Lo , Travis K. Warren , and Richard L. Mackman *
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Gilead Sciences, Inc., Foster City, California, USA.
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United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick,
Maryland, USA.
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UC San Diego, San Diego, California, USA.
4
Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
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ABSTRACT
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 (GSꢀ5734) with antiꢀEBOV EC =86 nM in
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macrophages as the clinical candidate. Structure activity relationships established that the 1′ꢀCN
group and Cꢀlinked nucleobase were critical for optimal antiꢀEBOV potency and selectivity
against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of
4b to enable preclinical efficacy in a nonhuman primate EBOV challenge model. Onceꢀdaily 10
mg/kg iv treatment on days 3ꢀ14 post infection had a significant effect on viremia and mortality,
resulting in 100% survival of infected treated animals [Nature 2016, 531, 381ꢀ385]. 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.
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INTRODUCTION
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 2013ꢀ2016 has been the largest recorded in history and presented an
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international public health emergency. Over 28,000 cases were confirmed in Guinea, Liberia,
and Sierra Leone resulting in >11,000 deaths including >500 healthcare workers, which severely
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strained the local medical infrastructure. In survivors, the Ebola virus (EBOV) can persist in
bodily fluids for months after the onset of acute infection potentially leading to EVDꢀrelated
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sequelae and viral recrudescence. While rare, secondary transmission has been documented to
4
occur through sexual intercourse implicating persistent virus in genital secretions. 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 singleꢀstranded, negativeꢀsense, nonꢀ
segmented 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
5
,6
associated with high mortality rates in subꢀSaharan Africa. Over the course of the recent West
African EVD outbreak, several direct acting antiꢀEbola agents including monoclonal antibodies
7
8ꢀ10
(
ZMapp™), interferingꢀRNAs , and small molecule nucleoside(tide) antivirals such as
1
1ꢀ13
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favipiravir (1),
and brincidofovir (2) have been evaluated in early clinical trials (Figure 1).
1
5
More recently another nucleoside analogue, galidesivir (3, BCX4430 ), has entered clinical
development. These developments are encouraging but to date, none of these potential
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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
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established.
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 analogs for the treatment of selected emerging viruses. A library of ~
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000 diverse nucleoside and nucleoside phosphonate analogs was harnessed from over two
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
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that contained the single Sp isomer 4b (GSꢀ5734 ) that was selected for development. This
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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 nonhuman primate
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(NHP) EVD challenge model. These data have been recently reported and will be summarized
along with the early clinical experience with 4b.
RESULTS AND DISCUSSION
The assembly of the ~1000 compound nucleos(t)ide screening library was heavily
focused toward ribose analogs 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 mosquitoꢀborne 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
riboseꢀlike’ core. These nucleosides were also predominantly Nꢀnucleosides. Less than 10% of
‘
the library comprised nucleoside phosphonates or acyclic analogs 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
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with endogenous natural nucleotide pools for incorporation into the replicating viral RNA. The
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first phosphorylation step to generate the nucleoside monophosphate is often rate limiting and
therefore the application of monophosphate prodrugs, especially phosphoramidates
TM
(
ProTides ), has been extensively explored in nucleoside analogs to bypass this initial
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phosphorylation step. A notable example includes the phosphoramidate prodrug Sofosbuvir (5)
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for the treatment of HCV (Figure 1). 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
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class is the phosphonamidate prodrug tenofovir alafenamide (6) for the treatment of HIV. 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
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nucleos(t)ides when screened in antiviral assays.
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
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BSLꢀ4 facility. From this screen nucleoside 4 , a 1′ꢀCN modified adenosine Cꢀnucleoside
emerged with submicromolar activity towards EBOV in Human Microvascular Endothelial Cells
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(
HMVECꢀTERT) cells (entry 2, Table 1). In addition, its phosphoramidate prodrug mixture 4a
(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 antiꢀEBOV 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.
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Table 1. SAR of nucleoside parents and selected prodrugs.
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EBOV EC50
HeLa cells
EBOV EC50
RSV EC50
HEpꢀ2 cells
(µM)
ꢀꢀ
HCV 1b EC50
Huhꢀ7 cells
(µM)
CC50
HEpꢀ2 cells
(µM)
CC50
Huhꢀ7 cells
(µM)
CC50
MT4 cells
(µM)
<0.01
>57
2
Entry Compound
HMVEC cells
(µM)
(µM)
ꢀꢀ
3
1
2
3
4
5
6
7
8
9
7
4
ꢀꢀ
0.0035
0.038
0.15
>20
0.17
0.10
ꢀꢀ
0.78
0.12
0.053
>10
ꢀꢀ
0.53
4.1
>100
9.2
>88
4
4
4a
4b
8
0.027
0.015
5.5
0.023
17
2.0
0.057
38
6.1
36
1.7
93
62
4.5
9
ꢀꢀ
>200
1.1
>88
6.9
>200
>100
>100
>100
>100
ꢀꢀ
>88
>44
>88
>44
>44
>44
>44
>44
51
120
9a
10
11
11a
12
13
13a
13b
ꢀꢀ
3.9
56
>32
ꢀꢀ
>100
7.3
>44
12
>53
>50
>20
ꢀꢀ
>10
ꢀꢀ
>57
1
0
1
2
3
4
63
2.5
53
3
3
1
1
1
1
ꢀꢀ
>100
>44
>44
0.37
0.31
32
50
>10
>100
>50
>100
>50
95
>57
1.4
7.8
4
27
13
>20
40
>20
1
2
Data reported is at least n≥2 in 384 well assay format unless otherwise noted. HMVEC cells = TERTꢀimmortalized human foreskin
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4
microvascular endothelial cells (ATCCꢀ4025) cells. 96 well assay format. n=1 data only.
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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 (CC <0.01 – 0.15 µM)
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2
4
associated with the unmodified Cꢀnucleoside 7 (entry 1, Table 1). 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 HEpꢀ2 and Huhꢀ7 cell lines. The prodrug mixture
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4
a in addition to potent antiꢀEBOV activity, demonstrated significant activity toward RSV and
HCV, with potencies similar or better than that for EBOV (EC <120 nM). The broad and potent
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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
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MERS and Junin viruses, and to a lesser extent Lassa. The antiviral selectivity of 4b toward
EBOV was 17ꢀ32 fold compared to the MT4 cell line CC , and higher in the other cell types
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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 analog 8
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2
(
entry 5) was less active toward EBOV and also displayed a higher degree of toxicity compared
to the 1′ꢀCN analog 4 illustrating how small changes in the polarity and size of the 1′ substituent
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2
can impact the overall profile. The 1′ꢀethynyl analog 9 (entry 6) and its corresponding 2ꢀ
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 Cꢀnucleoside analog which provides chemical and enzymatic stability
toward deglycosylation reactions at the anomeric center. However, alternate base modifications
including Nꢀnucleosides were also studied. Interestingly, the corresponding 1′ꢀCN modified
2
5
adenosine Nꢀanalog 10 (entry 8) was significantly less active toward all viruses, whilst the 1′ꢀ
CN modified Nꢀnucleoside pyrimidine 11 (entry 9) retained weak antiviral activity only for RSV
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and HCV.
The phosphoramidate prodrug of 1′ꢀCN cytidine 11a (entry 10) 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 was in
contrast to the trends uncovered with 2′ modifications. The 2′ꢀdeoxyꢀ2′ꢀfluorine analog 12 (entry
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1) and the 2′ꢀβꢀmethyl analog 13 (entry 12) both lacked significant antiviral activity.
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However, the 2′ꢀβꢀmethyl phosphoramidate prodrugs, analogs 13a and 13b (entries 13 and 14)
respectively, were both potent toward HCV, and only weakly active/inactive toward EBOV and
RSV. This result suggests that HCV polymerase is more able to accommodate the 2′ꢀβꢀmethyl
group compared to the EBOV and RSV polymerases. Taken together with the 1′ substitution and
nucleobase SAR, the EBOV and RSV polymerases demonstrated similar activity trends, whilst
HCV polymerase was differentiated in SAR at the 2′ position.
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To interrogate the cell based SAR more rigorously the active NTP metabolite 4tp was
tested toward the viral polymerases (Table 2). The triphosphate 4tp demonstrated a halfꢀmaximal
inhibitory concentration (IC ) of 1.1 µM against the RSV RdRp and 5.0 µM against HCV
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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
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concentration, and then calculate the NTP levels at the EC measured in the same cells. For
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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 NTP levels is not
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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 NTP. The
average NTP levels over the 72 h incubation of 4a were then used along with an average cell
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volume of 2 pL to calculate an estimated halfꢀmaximal 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 EC data demonstrated for the prodrug
50
mixture 4a across the three viruses when allowing for cell differences (Table 1). The selective
inhibition of the viral polymerases versus host polymerases is considered a key factor in the
30,31
development of a safe and effective nucleoside antiviral.
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.
NH2
N
O
P
O
P
O
P
N
O
O
O
O
N
O
O
O
O
N
HO
OH
4tp
4tp
4tp
Enzyme
1
IC50 (µM)
SNI rate (%)
RSV RdRp
HCV RdRp
1.1
5
ꢀ
ꢀ
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6
POLRMT
>200
>200
6
ꢀ
RNA Pol II
DNA Pol α
>200
>200
>200
ꢀ
ꢀ
DNA Pol β
0
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0
DNA Pol γ
0
1
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
3
2
polymerases, together with an analysis of the respective sequences.
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 analog retains antiviral potency across these viruses and
1
7
others suggesting that a pocket exists to accommodate the 1′ꢀCN group in many viral
3
3
polymerases including other filovirus. The 2′ꢀβꢀH of 4tp is in close proximity to E709 and
replacement of this group with a 2′ꢀβꢀmethyl (13tp) would be anticipated to interfere with E709
3
4
(Figure 2b). However, the 2′ꢀβꢀmethyl 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′ꢀβꢀmethyl
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 (IC >30 µM)
5
0
toward RSV.
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Figure 2. (a) Compound 4tp modeled into 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′ꢀβꢀH position of the ribose, and
corresponds to S282 in HCV; (b) Compound 13tp modeled into the EBOV polymerase active
site. The 2′ꢀβꢀmethyl 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′ꢀβꢀmethyl can be accommodated.
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NH2
NH2
N
N
O
P
O
P
O
P
O
P
O
P
O
P
N
N
O
O
O
O
O
O
O
O
N
N
O
O
O
O
O
O
O
O
N
N
HO
OH
HO
OH
4tp
13tp
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 diastereoisomers. The first generation
synthesis of 4 and the single Sp phosphoramidate prodrug 4b commenced with a glycosylation
reaction via metalꢀhalogen exchange of the bromoꢀbase 15 followed by addition into the
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ribolactone 14 (Scheme 1). Two conditions were identified to render this desired CꢀC bond
formation. The first condition (a) proceeded through addition of excess nꢀBuLi to a mixture of
TMSCl and 15, which was designed to result in lithiumꢀhalogen exchange after removal of the
acidic 6N protons by silyl protection. Addition of this in situ generated reagent to the
0
1
2
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9
0
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9
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1
2
3
4
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9
0
2
3,35,36
ribolactone 14 then afforded 16 in 25% yield.
The alternative conditions (b) employed
sodium hydride and 1,2ꢀbis(chlorodimethylsilyl)ethane for the 6N protection step, followed by
2
2,36
lithiumꢀhalogen exchange, and addition to the lactone to afford 16 in 60% yield.
a
Scheme 1. First generation synthesis of 4a.
a
Reagents and conditions: (a) nꢀBuLi, TMSCl, THF, −78 °C, 25%; (b) 1,2ꢀbis(chlorodimethylsilyl)ethane, NaH, nꢀ
BuLi, THF, −78 °C, 60%; (c) TMSCN, BF •Et O, CH Cl , −78 °C, 58% (89:11βꢀ17/α). (d) BCl , CH Cl , −78 °C,
3
2
2
2
3
2
2
7
4%. (e) 19, NMI, OP(OMe) , 21%; (f) OP(OPh)Cl , Et N, CH Cl , 0 °C, 23%.
3 2 3 2 2
The efficiency of both conditions was suboptimal as the yields were capricious and
highly dependent on the cryogenic temperatures and the rate of nꢀBuLi addition required for the
transformation. Furthermore, premature quenching and reduction of lithio base was observed,
which was rationalized to be a consequence of deprotonation alpha to the lactone under the
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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, βꢀanomer 17, by
3
7
chromatography. Following removal of the three benzyl protecting groups to afford 4, the
diastereomeric mixture of the phosphoramidoyl chloridate prodrug moiety 19, was then coupled
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2
3
to provide 4a in 21% yield, as an ~1:1 diastereomeric mixture. The two diastereomers were
3
8
resolved using chiral HPLC to afford the Sp isomer 4b and Rp isomer 4c respectively. 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.
a
Scheme 2. Second generation synthesis of 4b.
a
Reagents and conditions: (a) TMSCl, PhMgCl, iꢀPrMgCl•LiCl, THF, −20 °C, 40%; (b) TMSCN, TfOH, TMSOTf,
CH Cl , −78 °C, 85%; (c) BCl , CH Cl , −20 °C, 86%; (d) 2,2ꢀdimethoxypropane, H SO , acetone, rt, 90%; (e)
2
2
3
2
2
2
4
2
2b, MgCl , (iꢀPr) NEt, MeCN, 50 °C, 70%; (f) 37% HCl, THF, rt, 69%. (g) OP(OPh)Cl , Et N, CH Cl , −78 °C,
2 2 2 3 2 2
then 4ꢀnitrophenol, Et N, 0 °C, 80%; (h) iꢀPr O, 39%.
3
2
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
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17
studies (Scheme 2). The glycosylation step employed the iodoꢀbase 20 instead of the bromo
base which enabled a more facile metalꢀhalogen exchange compatible with iꢀPrMgCl•LiCl
39
complex. Treatment with PhMgCl and TMSCl provided 6N protection to remove the acidic
protons with a higher degree of control, and addition of iꢀPrMgCl•LiCl followed by the
ribolactone 14 at −20 °C afforded the glycosylation product 16 in a 40% yield. The milder
reagents and temperature enabled largeꢀscale 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 βꢀanomer. Benzyl deprotection was effected through treatment with BCl3
and 4 was readily isolated through crystallization. Acetonide protection of the 2′,3′ꢀhydroxyl
moeities with 2,2ꢀdimethoxypropane in the presence of H SO afforded 21 in 90% yield.
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
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9
0
2
4
Utilizing the 2′,3′ꢀacetonide protection was found to be optimal as the yield of the coupling
reaction with the pꢀnitrophenolate 2ꢀethylbutylꢀ ꢀalaninate prodrug mixture 22a was
L
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 pꢀnitrophenolate prodrug
precursor 22b in the presence of MgCl and Hunig’s base efficiently appended the prodrug group
2
in 70% yield as a single Sp isomer. Final deprotection of the acetonide with concentrated HCl in
THF afforded 4b in 69% yield.
The single Sp isomer 22b of the pꢀnitrophenolate 2ꢀethylbutylꢀLꢀalaninate prodrug
precursor 22a was prepared through a sequence beginning with exposure of 2ꢀethylbutylꢀLꢀ
alanine 18a to OP(OPh)Cl , followed by 4ꢀnitrophenol, to afford 22a as a diastereomeric mixture
2
at phosphorus. Importantly, the single Sp isomer 22b was readily resolved from the mixture in
3
9% yield through crystallization in diisopropyl ether, a discovery that was paramount for the
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success of the diastereoselective synthesis of the 4b. Thus, utilizing the pꢀnitrophenolate 2ꢀ
ethylbutylꢀLꢀalaninate 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 >200g was rapidly
prepared to support preclinical efficacy and toxicity studies.
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The stereochemistry of the pꢀnitrophenolate 2ꢀethylbutylꢀLꢀalaninate prodrug 22b and
candidate compound 4b were unambiguously assigned by small molecule XꢀRay crystallography
(Figure 3). In both cases the Sp isomer was established and suggests that the coupling with the
nucleoside and reagent follows a S 2 type inversion of the phosphorus stereocenter.
N
Figure 3. Thermal ellipsoid representations of (a) 22b and (b) 4b.
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0
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 tꢀBuMgCl or Lewis acidic conditions with MgCl in
2
polar aprotic solvents to afford the desired prodrugs in yields ranging from 10ꢀ43%. The
coupling reaction of the 2′,3′ꢀacetonide protected analogue 21 under lewis acidic conditions
followed by inꢀsitu acetonide deprotection in general afforded much higher yields ranging from
6
0ꢀ70% (analogue 4b and 4n). Both the paraꢀnitrophenol (PNP) and pentaꢀfluorophenol (PFP)
prodrug electrophiles were compatible in the coupling reactions and typically achieved
comparable yields. The reactions utilizing diastereomeric mixtures of the prodrug electrophiles,
2
2a, dꢀh, j, l, n provided the prodrug products in 1.5ꢀ2.6 to 1 diastereomeric ratios (unassigned)
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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.
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14
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16
17
18
19
20
21
22
23
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Scheme 3. Prodrug Synthesis.
X_______
Coupling
1
3
Prodrug
LG
Conditions
Product Yield
Isomer Ratio
2
Ester R
aa
Partner
22a
2ꢀEtBu
2ꢀEtBu
Et
LꢀAla PNP
LꢀAla PNP
LꢀPhe PNP
LꢀVal PNP
4
21
4
tꢀBuMgCl, THF, DMF, rt
4a
4b
4d
4e
4f
43%
69%
34%
43%
25%
24%
37%
38%
32%
43%
42%
10%
2.5 : 1
Sp Isomer
2.6 : 1
4
2
2
2
2
2b
2d
2e
2f
2 2
MgCl , (iꢀPr) NEt, THF, 50 °C; conc. HCl, 0 °C
tꢀBuMgCl, THF, DMF, rt
tꢀBuMgCl, THF, NMP, rt
tꢀBuMgCl, THF, NMP, 50 °C
tꢀBuMgCl, THF, NMP, rt
Et
4
1.8 : 1
Et
AIB
PNP
4
1.5 : 1
22g
Et
LꢀAla PNP
LꢀAla PNP
LꢀAla PNP
LꢀAla PNP
LꢀAla PFP
LꢀAla PNP
LꢀAla PFP
4
4g
4h
4i
2.5 :1
2
2
2h
cꢀBu
iꢀPr
4
MgCl
tꢀBuMgCl, THF, NMP, 50 °C
MgCl , (iꢀPr) NEt, DMF, 50 °C
tꢀBuMgCl, THF, DMF, rt
MgCl , (iꢀPr) NEt, DMF, 50 °C
tꢀBuMgCl, THF, DMF, rt
2
, (iꢀPr)
2
NEt, DMF, 50 °C
3 : 2
4
2i
4
Sp Isomer
1.5 : 1
22j
tꢀBu
cꢀPent
3ꢀPent
Neopent
4
2
2
4j
5
2
2k
4
4k
4l
Single Isomer
1.5 : 1
22l
4
2
2
5
2
2m
4
4m
Single Isomer
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6
2
1
2n
8b
8a
2ꢀEtBu
Et
DꢀAla PNP
21
4
MgCl
2
, (iꢀPr)
2
NEt, THF, 50 °C; conc. HCl, 0 °C
4n
4o
4p
66%
16%
58%
1.1 : 1
NA
LꢀAla
LꢀAla
NA
NA
POCl
3
, PO(OMe)
3
3
, Et
, Et
3
3
N, rt
N, rt
1
2ꢀEtBu
4
POCl
3
, PO(OMe)
NA
1
2
Prodrug is an undetermined mixture of diastereoisomers unless otherwise indicated. aa = amino acid, Ala = Alanine, Phe = Phenylalanine, AIB
=
2ꢀaminoisobutyrate, cꢀBu = cyclobutyl, cꢀPent = cyclopentyl, Pent = pentyl, Neopent = neopentyl, 2ꢀEtBu = 2ꢀethylbutyl, PNP = paraꢀ
0
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0
3
4
5
nitrophenolate, PFP = pentafluorophenolate. LG = Leaving Group. Single Sp isomer. Reagent was a single unassigned isomer at phosphorus.
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
monoꢀphosphate group enabling facile passive penetration into the cell. The prodrug breakdown
is initiated by intracellular esterases (e.g. carboxy esterase 1, cathepsin A) that cleave the ester
unraveling the carboxylate moiety, which then continues to breakdown to the monophosphate
1
7
that serves as the prescursor to synthesis of the intracellular NTP.
Prodrugs 4aꢀp 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 2ꢀ5) established that the
phenylalanine and alanine amino acids were the most promising (Table 1). 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. Nonꢀproximally 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. iꢀPr, tꢀBu, and 3ꢀpentyl (entries 7,8 and
1
0 respectively) were generally less active, consistent with the increased steric hindrance that
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likely slows down the cleavage rate by esterases. For example, the proximally branched 3ꢀpentyl
ester 4l (entry 10) has comparable log D to that of the neopentyl analog 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
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cell assay was reduced. The
corresponding ꢀAla analog mixture (entry 12) and the two bisphosphoramide prodrugs (entries
6 and 17) also had reduced activity compared to their monoamidate counterparts (entries 5 and
DꢀAla 2ꢀEtBu mixture (entry 15) was less potent that the
L
1
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 2ꢀEtBu ester mixture 4a. The Sp and Rp isomers of 4a (entries 14 and 15
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 (t > 60 min) was maintained to
½
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.
1
Table 3. Antiviral activity of prodrugs 4aꢀp.
EBOV
EC50
EBOV
EC50
EBOV
EC50
Human
Plasma
CC50
MT4
2
Entry
Compound
Ester
aa
LogD
3
4
HeLa
HMVEC
Macro
(nM)
t
1/2
(µM)
(nM)
(nM)
(min)
ꢀ
1
2
4
ꢀ
ꢀ
>20000
4380
780
>20000
270
>57
15
0.3
1.6
4d
Et
LꢀPhe
587
1584
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1
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2
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2
2
2
2
2
2
2
3
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3
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3
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5
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5
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3
4
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6
7
8
9
4e
4f
Et
Et
LꢀVal
AIB
7040
8470
2425
420
3151
1585
636
88
ꢀ
>100
>100
13
1584
1584
1584
815
1561
1584
1578
860
700
195
234
69
1.2
0.9
0.6
1.1
1.1
1.3
1.3
1.6
1.7
2.1
2.0
2.1
2.1
<0.3
2.7
ꢀ
ꢀ
4g
4h
4i
Et
LꢀAla
LꢀAla
LꢀAla
LꢀAla
LꢀAla
LꢀAla
cꢀBu
iꢀPr
ꢀ
6
1845
30410
633
367
3790
160
845
92
297
ꢀ
21
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4j
tꢀBu
cꢀPent
3ꢀPent
>100
8.8
>100
3
4k
4l
120
ꢀ
1
0
1
2
1810
168
1
1
4m
4a
4c
4b
4n
4o
4p
Neopent LꢀAla
ꢀ
2ꢀEtBu
2ꢀEtBu
2ꢀEtBu
2ꢀEtBu
Et
LꢀAla
LꢀAla
LꢀAla
DꢀAla
LꢀAla
LꢀAla
170
121
53
100
111
86
729
ꢀ
2
13
80
3
1
4
100
53
1.7
42
15
550
518
9102
678
1584
ꢀ
1
1
6
7
20420
970
>53
9
2ꢀEtBu
ꢀ
507
1
2
Data is at least n ≥2 unless otherwise reported. aa= Amino Acid, Ala = Alanine, Phe = Phenylalanine, AIB = 2ꢀaminoisobutyrate, cꢀBu =
3
cyclobutyl, cꢀPent = cyclopentyl, Pent = pentyl, 2ꢀEtBu = 2ꢀethylbutyl. HMVEC = TERTꢀimmortalized human foreskin microvascular
4
endothelial cells (ATCCꢀ4025), 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
4
1
of high expression of plasma carboxylesterases 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 gasterointestinal 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).
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Figure 4. Concentrationꢀtime 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
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4
b (black circle) and parent nucleoside 4 (blue triangle). (b) Intracellular concentration of active
metabolite 4tp in PBMCs (green diamond) and estimated 4tp EBOV IC = 5 µM (dashed black
5
0
line).
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 IC of 5 µM for EBOV, with a halfꢀlife of 14 h similar to that measured in vitro in
5
0
human macrophages. The long intracellular halfꢀlife of 4tp supported once daily iv
administration in the rhesus efficacy study and in the clinical program.
Preclinical in vivo efficacy for 4b was conducted in a EBOVꢀinfected 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
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9
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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17
have recently been published in full and will only be summarized here. The starting dose of 3
mg/kg iv was modeled based on the NTP 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 3mg/kg dose would
produce NTP levels that exceed the estimated IC for most of the dosing interval. Compound 4b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
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0
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0
1
2
3
4
5
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7
8
9
0
5
0
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 post infection was
improved. Animals administered 3 mg/kg 4b starting at day 0, had a survival rate of 33% whilst
those initiated on day 3 had a 66% survival at 28 days. This encouraging data was 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 eleven 3 mg/kg daily maintenance doses. All the animals in the two arms in which
4
b 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 x 10e4 RNA copies mLꢀ1) in 4 of the 6
animals on days 5 and 7 relative to the vehicleꢀtreated control in which the geometric mean
exceeded 10e9 copies mLꢀ1 at these time points. This data established the effectiveness of 4b in
treatment of EVD in NHPs and accelerated its progress into clinical development. In addition to
14
the efficacy studies, distribution studies in cynomolgus monkeys using [ C]4b at the same
effective dose of 10 mg/kg established the presence of drug related products in the potential
1
7
sanctuary sites for the virus including testes, epididymis, eyes and brain. At 4 h post dose, the
drug levels in the testes and epididymis exceeded those observed in plasma and even at 168 h
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post dose 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 4h, but were detectable
above the plasma levels at 168 h indicating a long halfꢀlife 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.
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Safety and pharmacokinetics of 4b administered as onceꢀdaily 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
4
2
had relapsed with symptoms of acute meningoencephalytis. 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 2
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
4
3
antibodies, blood transfusion and subsequently 4b. 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
4
transmission via virus in genital secretions. This has prompted the initiation of randomized,
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blinded, placeboꢀcontrolled phase 2 study (PREVAIL IV) which plans to enroll at least 60 adult
male survivors to receive either 100 mg 4b or placebo onceꢀdaily over 5 days to assess the effect
4
4
of 4b therapy on the viral shedding in semen. The results of this study could provide the first
evidence as to the potential of 4b to reduce virus replication in humans.
0
1
2
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9
0
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0
1
2
3
4
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8
9
0
CONCLUSION
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 halfꢀlife
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.
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EXPERIMENTAL
All organic compounds were synthesized at Gilead Sciences, Inc (Foster City, CA) 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 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
10
11
12
13
14
15
16
17
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21
22
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42
43
44
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
and are referenced from the residual protium in the NMR solvent (chloroformꢀd : δ 7.26,
1
methanolꢀd : δ 3.31, DMSOꢀd : δ 2.50). Data is reported as follows: chemical shift [multiplicity
4
6
(s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sep = septet, m = multiplet, br =
broad, app = apparent), coupling constants (J) in Hertz, integration. Carbonꢀ13 nuclear magnetic
resonance spectra are reported in parts per million on the δ scale and are referenced from the
carbon resonances of the solvent (chloroformꢀd : δ 77.16, methanolꢀd : δ 49.15, DMSOꢀd : δ
1
4
6
39.52). Data is reported as follows: chemical shift. No special nomenclature is used for
equivalent carbons. Phosphorusꢀ31 nuclear magnetic resonance spectra are reported in parts per
million on the δ scale. Data is 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 glassplates 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 highꢀ
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 100Å, 100 × 4.6 mm column; 2ꢀ98% gradient of 0.1% trifluoroacetic acid
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2
2
2
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4
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5
5
5
5
5
6
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. Highꢀresolution 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 mm column at 30 °C, with a 10ꢀ90% gradient of 0.05% trifluoroacetic
acid in water and 0.05% trifluoroacetic acid in acetonitrile. LCꢀMS was conducted on a Thermo
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
–
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 u C18 110A column (30
mm × 4.60 mm), eluting with 0.05% formic acid in 1% acetonitrile/waterand 0.05% formic acid
in 99% acetonitrile/water. Optical rotations were recorded on a Jasco Pꢀ2000 Polarimeter.
The synthesis, characterization data, and associated references for the following
compounds are provided in supporting information: 4, 4a, 7ꢀ11, 9a, 11a, 12ꢀ13, 13a, 13b, 4tp,
13tp, 16ꢀ17, 18a, 19, 21, and 22aꢀn.
(S)ꢀ2ꢀethylbutyl 2ꢀ(((S)ꢀ(((2R,3S,4R,5R)ꢀ5ꢀ(4ꢀaminopyrrolo[2,1ꢀf][1,2,4]triazinꢀ7ꢀyl)ꢀ5ꢀ
cyanoꢀ3,4ꢀdihydroxytetrahydrofuranꢀ2ꢀ
yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4b). Compound 4b was prepared from
1
7 1
4
and 22b as described previously.
H NMR (400 MHz, methanolꢀd ): δ 7.86 (s, 1H), 7.33 –
4
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
0
1
.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),
.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),
1
3
.85 (t, J = 7.4 Hz, 6H). CꢀNMR (100 MHz, methanolꢀd ): δ 174.98, 174.92, 157.18, 152.14,
4
52.07, 148.27, 130.68, 126.04, 125.51, 121.33, 121.28, 117.90, 117.58, 112.29, 102.60, 84.31,
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4.22, 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.;
3
1
+
PꢀNMR (162 MHz, methanolꢀd ): δ 3.66 (s); HRMS (m/z): [M] calcd for C H N O P,
4
27 35
6
8
2
1
6
02.2254; found, 602.2274. [α] –21 (c 1.0, MeOH).
D
10
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(
S)ꢀ2ꢀethylbutyl 2ꢀ(((R)ꢀ(((2R,3S,4R,5R)ꢀ5ꢀ(4ꢀaminopyrrolo[2,1ꢀf][1,2,4]triazinꢀ7ꢀyl)ꢀ5ꢀ
cyanoꢀ3,4ꢀdihydroxytetrahydrofuranꢀ2ꢀ
yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (4c). Compound 4c was prepared from
4
a by chiral chromatography. 4a was dissolved in acetonitrile. The resulting solution was loaded
onto Lux Celluloseꢀ2 chiral column, equilibrated in acetonitrile, and eluted with isocratic
acetonitrile/methanol (95:5 vol/vol). The first eluting compound was the Rp diastereomer 4c, and
1
the second eluting compound was the Sp diastereomer 4b. H NMR (400 MHz, methanolꢀd4)
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 =
=
3
1
7
.3 Hz, 4H), 1.29 (d, J = 7.2 Hz, 3H), 0.87 (t, J = 7.4 Hz, 6H). P NMR (162 MHz, methanolꢀd )
4
21
δ 3.71 (s). MS m/z 603.1 [M+1]. [α] –46 (c 1.0, MeOH).
D
(2S)ꢀethylꢀ2ꢀ(((((2R,3S,4R,5R)ꢀ5ꢀ(4ꢀaminopyrrolo[2,1ꢀf][1,2,4]triazinꢀ7ꢀyl)ꢀ5ꢀcyanoꢀ3,4ꢀ
dihydroxytetrahydrofuranꢀ2ꢀyl)methoxy)(phenoxy)phosphoryl)amino)ꢀ3ꢀ
phenylpropanoate (4d). Compound 4 (0.030 g, 0.103 mmol) was dissolved in DMF (1 mL) and
then THF (0.5 mL) was added. tꢀBuMgCl (1M/THF, 154.5 ꢁL, 0.154 ꢁmol) was added to the
reaction in a dropꢀwise 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
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