Synthesis and Biological Evaluation of (E)-4-Hydroxy-3-methylbut-2-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate Prodrugs as Activators of Vγ9/Vδ2 T-Cell Immune Responses

Article abstract of DOI:10.1021/acs.jmedchem.7b01824

The aryloxy triester phosphoramidate prodrug approach has been used with success in drug discovery. Herein, we describe the first application of this prodrug technology to the monophosphate derivative of the phosphoantigen HMBPP and one of its analogues. Some of these prodrugs exhibited specific and potent activation of Vγ9/Vδ2 T-cells, which were then able to lyse bladder cancer cells in vitro. This work highlights the promise of this prodrug technology in the discovery of novel immunotherapeutics.

Full text of DOI:10.1021/acs.jmedchem.7b01824

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Brief Article
Synthesis and Biological Evaluation of (E)-4-Hydroxy-3-Methylbut-2-
enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate
Prodrugs as Activators of V#9/V#2 T-Cells Immune Response
Martin S. Davey, Roshni Malde, Rory C Mykura, Alfie T Baker, Taher
E Taher, Cécile S. Le Duff, Benjamin E. Willcox, and Youcef Mehellou
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Feb 2018
Downloaded from http://pubs.acs.org on February 19, 2018
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Synthesis and Biological Evaluation of (E)-4-Hydroxy-3-Methylbut-
2
-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate
Prdrugs as Activators of Vγ9/Vδ2 T-Cells Immune Response
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a,‡
b,‡
b,‡
a
a
Martin S. Davey, Roshni Malde, Rory C. Mykura, Alfie T. Baker, Taher E. Taher, Cécile S. Le
b
*,a
*,c
Duff, Benjamin E. Willcox, Youcef Mehellou
aCancer Immunology and Immunotherapy Centre, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
bSchool of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
cSchool of Pharmacy and Pharmaceutical Sciences. Redwood Building, Cardiff University, Cardiff, CF10 3NB, UK.
Immunotherapy, T-cell, Phosphoantigen, Prodrug, ProPAgen.
ABSTRACT: The aryloxy triester phosphoramidate prodrug approach has been used with success in drug discovery. Herein, we
describe the first application of this prodrug technology to the monophosphate derivative of the phosphoantigen HMBPP and one of
its analogues. Some of these prodrugs exhibited specific and potent activation of Vγ9/Vδ2 T-cells, which were then able to lyse
bladder cancer cells in vitro. This work highlights the promise of this prodrug technology in the discovery of novel immunotherapeu-
tics.
INTRODUCTION
protein, there is an increasing body of evidence that supports
the notion that these PAgs bind the intracellular B30.2 domain
of butyrophilin 3A1.
1
Present since birth, Vγ9/Vδ2 T-cells represent the dominant
subtype of human γδ T-cells in adult peripheral blood. They
10, 12-15
2
expand in response to various infections, including tuberculo-
sis, leprosy, typhoid, malaria, and toxoplasmosis, and studies in
primate models have suggested a role in immunity to Mycobac-
Encouraged by Vγ9/Vδ2 T-cells’ ability to mount immune
responses towards pathogens, lyse tumor cells, as well as their
amenability to be targeted and modulated by small molecules
(PAgs and their synthetic mimics), we embarked on the discov-
ery of small molecules that have the potential to activate
Vγ9/Vδ2 T-cells. Given our interest in the discovery and devel-
opment of phosphorylated molecules and their prodrugs as ther-
3
terium tuberculosis. Interestingly, they also exhibited an abil-
2
ity to target and lyse a diverse range of cancer cells in vitro.
Such properties have made the Vγ9/Vδ2 subset a major focus
4
in the therapeutic exploitation of γδ T-cells.
16-18
apeutics, we focused our efforts on the natural phosphoanti-
Interestingly, Vγ9/Vδ2 T-cells have been shown to be acti-
vated by small molecule phosphoantigens (PAg) such as (E)-4-
hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and
gen HMBPP as it is the most potent activator of Vγ9/Vδ2 T-
19
cells reported to date (Figure 1). HMBPP has a pyrophosphate
5, 6
group, which accounts for poor drug-like properties namely
poor cell membrane permeability, due its charged nature under
physiological conditions (pH ≤ 7.4) and limited in vivo stabil-
ity. These properties have hindered the development of many
drugs with unmasked phosphate or pyrophosphate groups. To
overcome these drawbacks, numerous phosphate prodrug strat-
egies have been developed and used with success in the discov-
ery of mostly nucleotide monophosphates and monophospho-
isopentenyl pyrophosphate (IPP) (Figure 1). Beyond these
natural ligands, two synthetic molecules, Risedronate and
Zoledronate, activate Vγ9/Vδ2 T-cells through accumulation of
IPP and are currently used in the clinic to treat osteoporosis and
7
-9
some types of cancer (Figure 1).
20-22
nates.
There have been reports in the literature of the application of
the bis-pivaloyloxymethyl (bisPOM) phosphate prodrug tech-
nology and its derivatives to HMBP and its diphosphonates,
which resulted in potent phosphoantigens though these were
Figure 1. Chemical structures of natural phosphoantigens
HMBPP and IPP as well as synthetic molecules Risedronate
and Zoledronate, which activate Vγ9/Vδ2 T-cells.
19, 23-25
less potent than the parent phosphoantigen HMBPP.
Aim-
ing to discover phosphoantigen prodrugs that are as potent as
HMBPP, we focused our work on the aryloxy triester phospho-
ramidate prodrug approach, in which the monophosphate or
monophosphonate groups are masked by an aryl motif and an
amino acid ester moiety. This prodrug technology is known to
be more efficient in delivering monophosphorylated molecules
The mechanism by which these small molecule phosphoanti-
26
gens activate Vγ9/Vδ2 T-cells is understood to be mediated by
10, 11
the type-1 transmembrane protein butyrophilin 3A1.
Alt-
hough conflicting reports exist as to whether PAgs bind to the
extracellular or intracellular domains of this transmembrane
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than the bisPOM approach. Over the last decade or so, it has
led to at least ten clinical candidates with two being eventually
were synthesized following the procedure reported by Mehellou
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et al and as shown in Scheme 1b. These were coupled to 4 in
the presence of triethylamine or N-methylimidazole (NMI) to
afford the desired HMBP ProPAgens, 5a-d, in modest yields.
Notably, NMI was only used in cases when TEA gave very low
yields. The removal of the TBS protecting group was initially
2
8
approved for clinical use. Notably, this prodrug approach has
mostly been used on nucleotide monophosphates and mono-
phosphonates. In this work, we applied this powerful phosphate
prodrug technology to the monophosphate derivative of the
phosphoantigen HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl
phosphate (HMBP). The aryloxy triester phosphoramidate pro-
drugs of the phosphoantigen HMBP will be referred to as
HMBP ProPAgens in this work.
30
pursued using TBAF in THF. However, this strategy did not
work in our hands as the reaction yielded so many products and
no traces of the desired HMBP ProPAgens were detected by
mass spectroscopy. As an alternative, we used mild acidic con-
ditions, 0.1 equivalents of 1.25 M HCl in methanol, and this
achieved the TBS deprotection without degrading the phos-
phate masking moieties to afford the desired HMBP ProPAgens
6a-d in good yields.
RESULTS AND DISCUSSION
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The synthesis of HMBP ProPAgens was similar to that re-
2
9
ported by Reichenberg et al. Briefly, the hydroxyl group of 1-
hydroxypropan-2-one (1) was protected with TBS in the pres-
ence of imidazole (Scheme 1a). The ether product 2 was then
treated with a Horner-Wadsworth-Emmons reagent, specifi-
cally triethyl phosphonoacetate, to create the double bond with
E selectivity and yield the ester product 3. This compound, 3,
was produced in a mixture of E/Z isomers with a 5:1 ratio sim-
The unprotected HMBP ProPAgens 6a-d, however, exhibited
low stability and underwent rapid degradation, which prevented
their biological testing (purity < 95%). In order to get an insight
into the metabolism of ProPAgens 6a-d, compound 6d was in-
cubated with the carboxypeptidase cathepsin A in vitro and the
3
1
5
reaction was monitored with P-NMR. The data show that after
ilar to what is reported in the literature. The E/Z isomers were
separated by column chromatography using solvent mixture of
% to 7 % diethyl ether in hexane. The two products were then
evaluated with H and C NMR utilizing previous work by
72 h, the major metabolite had a phosphorous peak at ~ 1.95
ppm (Supporting Figure S1). Mass spectroscopy analysis of
5
1
13
these samples showed that the degradation product, which had
5
31
Hintz et al who performed NOSEY experiments to determine
a
P-NMR peak of ~ 1.95 ppm, was the phosphate group
masked with the aryl motif and the amino acid ester moiety
(Supporting Figure S2). This indicated that the P-O bond in
HMBP ProPAgens 6a-d was liable and was cleaved off to re-
lease the unphopshorylated PAg backbone rather than HMBP.
This may explain literature reports pursuing the phosphonates
the identity of each isomer. The desired E-isomer, 3, was sub-
sequently reduced using lithium aluminum hydride in THF to
afford the key intermediate 4. The coupling of compound 4 to
phosphorochloridates to afford the desired prodrugs was
1
7, 18
achieved using standard procedures.
Phosphorochloridates,
a.
2
of HMBP where the –O-P- bond was replaced by a –CH -P- one
O
O
19
a
b
OEt
and lack of reports on the native HMBP phosphate prodrugs.
OH
OTBS
TBSO
O
Interestingly, HMBP ProPAgens 5a-d, which had the side
chain hydroxyl group protected with a TBS moiety exhibited
better stability than compounds 6a-d and hence we were able to
characterize them fully and obtain a measure of their purity (see
Supporting Information). Prodrugs 5a-d were then investigated
for their ability to activate Vγ9/Vδ2 T-cells. For this, peripheral
blood mononuclear cells (PBMCs) containing Vγ9/Vδ2 T-cells
derived from healthy donors were incubated with increasing
concentrations of HMBPP, Zoledronate or HMBP ProPAgens
1
2
3, E:Z 5:1
O
O
P
O
d
TBSO
c
TBSO
HN
OH
R: 5a: Me; 5b: Bn;
c: iPr; 5d: tBu
O
O
R
4
5
5a-d (up to 100 µM) (Figure 2a and b). Peripheral blood γδ T-
e
f
cells lack appreciable levels of surface CD69 or CD25 under
steady state conditions, but T-cell receptor (TCR) stimulation
O
O
3
1
O
P
O
O
P
O
upregulates both T-cell activation markers. PAg responsive
Vγ9/Vδ2 T-cells were then distinguished by TCR Vγ9 and Vδ2
expression and assessed for the upregulation of CD69 and
CD25.
HO
HO
HN
O
HN
O
6
O
R
6
O
R
R: 6a: Me; 6b: Bn;
6
c: iPr; 6d: tBu
As shown in Figures 2c and d, the natural phosphoantigen
b.
O
HMBPP showed significant activation of Vγ9/Vδ2 T-cells,
O
Cl
H
g
O
P
Cl
19
EC50 = 0.06 nM, comparable to its reported potency. Addi-
O
HN
O
P
Cl
3
N
R
Cl
tionally, Zoledronate showed a moderate activation, EC50 ~ 500
nM as expected (Figures 2c and d). The four HMBP ProPAgens
O
O
O
R
7
8
R: 9a: Me; 9b: Bn;
c: iPr; 9d: tBu
5a-d exhibited potent Vγ9/Vδ2 T-cell activation with 5b being
9
the most potent, EC50 = 0.45 nM, followed by 5a, EC50 = 1.38
nM. Such potency makes HMBP ProPAgen 5b 1,081-times
more active than the clinically used agent Zoledronate. The
other two HMBP ProPAgens also exhibited low nanomolar ac-
tivation of Vγ9/Vδ2 T-cells, 10.62 nM for 5c and 8.92 nM for
5d (Figures 2c and d). Impressively, CD8+ ab T-cells, which
are activated by peptides, showed no reactivity towards HMBP
ProPAgens exemplifying their specificity towards Vγ9/Vδ2 T-
cells (Figure 2e). Notably, the unphopshorylated backbone
Scheme 1. A. Synthesis of HMBP ProPAgens. Reagents and con-
ditions: (a) TBSCl, imidazole, DCM, rt, yield 95%; (b) triethyl
phosphonoacetate, NaH, THF, 0 °C, yield 50%; (c) LiAlH
°C, yield 40%; (d) 9a-d, TEA or NMI, DCM, yields 36-56%;
e) TBAF, THF; (f) HCl, MeOH, yields 20-74%. B. Synthesis of
aryl phosphorochloridates. Reagents and conditions: (g) Et O,
4
, THF,
0
(
2
TEA, -78 °C, yields 54-95%. Me: methyl; iPr: isopropyl; tBu: tert-
butyl; Bn: benzyl.
9a-d, bearing methyl, isopropyl, tert-butyl or benzyl esters,
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Journal of Medicinal Chemistry
a.
+
HMBPP [10-2 µM]
73.5%
0
1
2
3
4
5
6
7
8
9
CD3 T-cells
Media
Zoledronate [10 µM]
39.2%
23.4%
0.00%
TCR Vγ9
CD25
b.
ProPAgen 5a [10-1 µM]
ProPAgen 5b [10-1 µM]
ProPAgen5c [10-1 µM]
ProPAgen 5d [10-1 µM]
29.9%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
4
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4
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
9.0%
50.6%
31.0%
CD25
c.1 0 0
HMBPP
1 0 0
Zoledronate
10 0
ProPAgen 5a
d.
EC50 (nM)
ED50 (µM)
SI
7
5
2
5
0
5
0
7 5
5 0
2 5
0
7 5
5 0
2 5
0
HMBPP
0.06
486.60
1.38
0.45
10.62
8.92
>100
>100
29.52
8.51
83.18
>100
-
-
Zoledronate
ProPAgen 5a
ProPAgen 5b
21,391
12,911
7,832
-
ProPAgen 5c
ProPAgen 5d
M -5 -4 -3 -2 -1
0
1
2
3
M -5 -4 -3 -2 -1
0
1
2
3
M -5 -4 -3 -2 -1
0
1
2 3
µM
e.
100
1
0 0
ProPAgen 5b
1 0 0
7 5
5 0
2 5
0
ProPAgen 5c
1 0 0
7 5
5 0
2 5
0
ProPAgen 5d
H M B -P P
Z o le d ro n a te
5 a
7
5
7
5
2
5
0
5
0
5
5
5
b
c
d
5 0
2 5
0
M -5 -4 -3 -2 -1
0
1
2
3
M -5 -4 -3 -2 -1
0
1
2
3
M -5 -4 -3 -2 -1
0
1
2
3
M
-5 -4 -3 -2 -1
0
1
2 3
µM
µM
+
Figure 2. Activation of human Vγ9/Vδ2 T cells by HMBP ProPAgens. (a) Human peripheral blood mononuclear cells (PBMC)
were incubated with media or indicated concentrations of HMB-PP or Zoledronate for 18 h. TCR Vγ9/Vδ2 T cells were then assessed
for the upregulation of cell surface markers, CD69 and CD25. Data representative of n = 5. (b). As in (a), with PBMC incubated with
indicated concentrations of HMBP ProPAgens. Data representative n = 4. (c). as in (a) and (b), with data showing titrations of each
of HMB-PP, Zoledronate and HMBP ProPAgens, alongside a medium control (M). Data from n = 4-5 donors. (d) EC50, ED50 and
selectivity index (SI) values for each HMBP ProPAgens. Specific cell death, for ED50 values, was calculated after adjusting for non-
+
+
+
specific cell death in medium controls. (e) CD69 and CD25 activation in CD3 CD8 αβ T-cells for each compound.
compound 4 did now show any activation of Vγ9/Vδ2 T-cells
benzyl esters, e.g. 5b, has higher lipophilicity and thus better
(passive) cellular uptake. Also, the benzyl group is better leav-
ing group than the other aliphatic esters and thus the metabolism
of the benzyl ester of 5b proceeds faster than the other
ProPAgens, 5a, 5c and 5d. Together, these explain the superior
activity of ProPAgen 5b. HMBP ProPAgen 5a also exhibited
relatively potent activation of Vγ9/Vδ2 T-cells with EC50 = 1.38
nM. The fact that ProPAgen 5c, which has an isopropyl ester,
exhibited lower potency in activating Vγ9/Vδ2 T-cells as com-
pared to 5a and 5d was surprising as often phosphoramidate
prodrugs with this ester exhibit similar activity to those with a
methyl ester. However, the less potent activity observed with
the HMBP ProPAgen 5d was expected and is in agreement with
the literature where phosphoramidate prodrugs with tBu esters
show lower biological activity than those with other ester mo-
tifs. This is because the hydrolysis of the tBu ester group of the
phosphoramidates by esterase enzymes proceeds much slower
than those with Me, iPr and Bn esters.
(data not shown).
Next, we tested the potential for ProPAgens to sensitise the
urinary bladder carcinoma cell line T24 for targeted killing by
in vitro expanded Vγ9/Vδ2 T cells. Without sensitization, me-
dium pulsed T24 cells were poorly targeted by increasing ratios
of Vγ9/Vδ2 T cells, but upon pulsing for four hours with
zoledronate or ProPAgens 5a-d, T24 cells were specifically
lysed (Figure 3a). The short pulsing period, poor lipophilicity
and requirement to target a metabolic enzyme resulted in only
a marginal increase in specific T24 cell killing by zoledronate
(
Figure 3b). In contrast, a 1000-fold lower concentration of each
ProPAgen mediated a 2-4-fold increase in specific lysis of T24
over the same period (Figure 3b).
The activity across the different HMBP ProPAgens in both
biological assays was in agreement with what is typically ob-
served with aryloxy triester phosphoramidate (ProTide) pro-
drugs of nucleoside analogues as it correlated with the lipo-
philicity and rate of degradation. The HMBP ProPAgen with
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
a flow rate of 20 mL/min. The purity of the tested compounds was
Page 4 of 7
a.
b.
5
0
6
5
0
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5
5
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6
Media
**
determined by high-performance liquid chromatography (HPLC)
where all of the tested compounds had ³ 95% purity.
1-((tert-butyldimethylsilyl)oxy)propan-2-one, 2. Hydroxyace-
tone 1 (1.00 g, 13.50 mmol, 1 eq.) and TBSCl (3.05 g, 20.25 mmol,
Zoledronate
ProPAgen 5a
ProPAgen 5b
40
30
*
*
*
4
3
0
0
*
ProPAgen 5c
ProPAgen 5d
2
0
1
.5 eq.) were combined in dry CH
Imidazole (2.02 g, 29.70, 2.2 eq.) was then added portion-wise at 0
C. The mixture was allowed to warm to room temperature and left
to stir at that temperature for 2.5 h. The reaction mixture was then
washed with brine (50 mL), extracted with Et O (50 mL x 3), dried
MgSO ) and the solvents removed under reduced pressure to give
a crude oil, which was purified via column chromatography (10:1
Hexane:EtOAc). 2 obtained as a colorless oil (2.45 g, 95%). δ
NMR (300 MHz, CDCl ): 4.15 (s, 2H, CH ), 2.17 (s, 3H, CH ),
), 0.09 (s, 6H, CH ). δ NMR (101 MHz, CDCl ):
09.25, 69.57, 25.98, 25.76, 18.29, -5.51. MS-ESI (%): 211.1 (100,
2 2
Cl (50 mL) under an argon.
2
1
0
0
10
0
°
0
1
:0
1:1
1:5
1:10
1:20
5
a
5b 5c 5d
10 nM]
Target: Efector ratio
Media
2
[
(
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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6
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. HMBP ProPAgens mediate the specific lysis of
bladder cancer cells by Vγ9/Vδ2 T-cells. (a) Human T24 uri-
nary bladder carcinoma cell lines (Target) were pulsed for 4 hours
with 10 nM of the indicated ProPAgens, media or Zoledronate, then
co-cultured for 18 hours with expanded Vγ9/Vδ2 T cells (Effector)
at the indicated Target:Effector ratios. Specific killing of target
cells was measured by an amine-reactive dead cell marker staining.
Data display the mean of n = 2. (b). As in (a) showing the mean ±
SD specific killing of T24 cells at 1:10 Target:Effector ratio for
each indicated treatments, n = 2. * P < 0.05; ** P < 0.01; deter-
mined by One-way ANOVA.
H
3
2
3
0.93 (s, 9H, CH
3
3
C
3
2
M+Na).
Ethyl (E)-4-((tert-butyldimethylsilyl)oxy)-3-methylbut-2-eno-
ate, 3. To a 0 °C suspension of NaH (0.20 g, 8.38 mmol, 1 eq.) in
dry THF (50 mL) under an argon was added diethyl phosphonoace-
tate (1.88 g, 8.38 mmol, 1 eq.) dropwise. The resulting solution was
left to stir for 25 min at 0 °C. Silyl ether 2 (1.5 g, 7.96 mmol, 0.95
eq.) was then added dropwise as a solution in dry THF (25 mL).
The mixture was then stirred at 0 °C for a further 30 min, then
warmed to room temperature and stirred for a further 2 h, or until
complete via TLC. A saturated solution of brine (50 mL) was
added, dropwise at first, then the organic layer was extracted with
CONCLUSION
We reported the application of the aryloxy triester phospho-
ramidate technology to the phosphoantigen HMBP and one of
its synthetic analogues to generate HMBP ProPAgens. Alt-
hough the ProPAgens of the native HMBP compound (6a-d)
were of very low stability, those of the synthetic analogue (5a-
d) were more stable. These later ones exhibited potent activa-
tion of Vγ9/Vδ2 T-cells. Notably, HMBP ProPAgen 5b was the
most potent activator of Vγ9/Vδ2 T-cells, EC50 = 0.45 nM with
the other three ProPAgens, 5a, c and d, also showing low nano-
molar activation. Impressively, Vγ9/Vδ2 T-cells activated by
HMBP ProPAgens exhibited potent lysis of urinary bladder car-
cinoma cancer cells (T24) in vitro. In terms of specificity,
HMBP ProPAgens, 5a-d, showed excellent specificity towards
the activation of Vγ9/Vδ2 T-cells as they had no effects of other
T-cells such as CD8+ αβ T-cells. Efforts aimed at improving
the stability of the native HMBP ProPAgens are currently un-
derway and will be reported in the future. In conclusion, the ap-
plication of the aryloxy triester phosphoramidate prodrug tech-
nology to HMBP to generate HMBP ProPAgens yielded potent
and specific activators of Vγ9/Vδ2 T-cells. Together, this show-
cases the promise of this prodrug technology in the discovery
of novel immunotherapeutic agents.
Et
to give a mixture of E:Z isomers (5:1 via H NMR) as a crude oil.
This was purified via column chromatography (10% Et O in Hex-
ane) to give product 3 as a colorless oil (1.04 g, 50%). δ NMR
(300 MHz, CDCl ): 5.99 (d, J = 1.4 Hz, 1H, CH), 4.17 (q, J = 7.1
Hz, 2H, CH ), 4.11 (d, J = 1.4 Hz, 2H, CH ), 2.05 (s (broad), 3H,
), 1.29 (t, J = 7.1 Hz, 3H, CH ), 0.93 (s, 9H, CH ), 0.09 (s, 6H,
). δ NMR (101 MHz, CDCl ): 167.10, 157.16, 113.35, 67.10,
2 4
O (50 mL x 3), dried (MgSO ) and the solvents were removed
1
2
H
3
2
2
CH
CH
3
3
3
3
C
3
5
2
9.59, 25.88, 18.37, 15.44, 14.36, -5.45. MS-ESI (%): 258.2 (8, M),
43.2 (100, M – CH ).
3
(E)-4-((tert-butyldimethylsilyl)oxy)-3-methylbut-2-en-1-ol, 4.
In a flame dried Schleck flask were placed ester 3 (0.5 g, 1.93
mmol, 1 eq.) and 20 mL dry THF, and the resulting mixture was
4
cooled to 0 °C with stirring. Then LiAlH (1 M solution in THF)
(
1.93 mL, 1.93 mmol, 1 eq.) was added dropwise, and the solution
was allowed to warm up to room temperature and stirred for 2 h.
MeOH (1 mL) was then added cautiously, after which a saturated
solution of sodium potassium tartrate (30 mL) was added which
formed a white gel. This was stirred overnight at room temperature
and then the phases were allowed to separate. The organic layer
was collected and the aqueous layer was washed with Et
2
O (20 mL
x 3), dried (MgSO ) and the solvents were removed under reduced
4
pressure to leave a crude oil. This was then purified by column
chromatography using hexane:ethyl acetate (4:1) as an eluant to
EXPERIMENTAL SECTION
give product 4 (0.1677 g, 40%) as a colorless oil. δ
CDCl ) 5.72-5.65 (m, 1H, CH), 4.21 (d, J = 6.9 Hz, 2H, CH
(s, 2H, CH ), 1.65 (s (broad), 3H, CH ), 0.92 (s, 9H, 3 x CH
). δ (101 MHz, CDCl
5.94, 18.41, 13.50, -5.34. MS (ESI) (%): 239.1 (100, M+Na).
H
(300 MHz,
General Information. All of the reactions were carried out under
argon atmosphere and were monitored with analytical thin layer
chromatography (TLC). NMR data were recorded either on a
Bruker AV300, AVIII300, AV400, AVIII400 or DRX500 spec-
trometers in the deuterated solvents indicated and the spectra were
calibrated on residual solvent peaks. Chemical shifts (δ) are quoted
in ppm and J values are quoted in Hz. In reporting spectral data, the
following abbreviations were used: s (singlet), d (doublet), t (tri-
plet), q (quartet), dd (doublet of doublets), td (triplet of doublets)
and m (multiplet). HPLC was carried out on a DIONEX summit
P580 quaternary low-pressure gradient pump with a built-in vac-
uum degasser using a Summit UVD 170s UV/Vis multichannel de-
tector. HPLC grade solvents were used. Chromeleon software was
used to visualize and process the obtained chromatograms. Analyt-
ical separations used a flow rate of 1 mL/min and preparative used
3
2
), 4.04
), 0.08
2
3
3
(
s, 6H, CH
3
C
3
) 138.32, 122.53, 67.66, 59.14,
2
Methyl ((((E)-4-((tert-butyldimethylsilyl)oxy)-3-methylbut-2-
en-1-yl)oxy)(phenoxy)phosphoryl)-L-alaninate, 5a. To a stirring
solution of alcohol 4 (0.1301 g, 0.6166 mmol, 1 eq.) in dry CH Cl
2 2
(10 mL) was added methyl phosphorochloridate 9a (0.2055 g,
0.7400 mmol, 1.2 eq.). The solution was cooled to 0 °C and NMI
(
0.10 mL g, 1.2333 mmol, 2 eq.) was added dropwise and the re-
sulting solution was warmed to room temperature and stirred over-
night. Brine (10 mL) was then added and the organic layer sepa-
rated off. The aqueous layer was then extracted with Et
x 3), the organic layers combined, dried (MgSO ) and the solvents
removed under reduced pressure to leave a crude oil. This was then
2
O (20 mL
4
ACS Paragon Plus Environment

Page 5 of 7
Journal of Medicinal Chemistry
67.27, 63.15 (t, J = 5.0 Hz), 50.66 (2 x s), 27.86, 25.87, 21.06 (2 x
purified by column chromatography (4:1 Hex:EtOAc to 1:1
Hex:EtOAc) to give compound 5a (0.1228 g, 43%) as a colourless
oil. δ NMR (400 MHz, CDCl ): 7.31 (t, J = 7.3 Hz, 2H, 2 x CH),
.21 (t, J = 7.3 Hz, 2H, 2 x CH), 7.14 (t, J = 7.3 Hz, 1H, CH), 5.75-
.59 (m, 1H, CH), 4.72-4.64 (m, 2H, CH ), 4.10-3.97 (m, 3H, CH
CH), 3.70 (d, J = 8.8 Hz, 3H, CH ), 3.53 (dd, J = 18.0, 9.7 Hz,
H, NH), 1.65 (d, J = 3.5 Hz, 3H, CH ), 1.37 (t, J = 7.4 Hz, 3H,
CH ), 0.91 (s, 9H, 3 x CH ), 0.07 (s, 6H, 2 x CH ). δ NMR (101
MHz, CDCl ): 173.96 (d, J = 6.0 Hz), 150.89 (d, J = 5.7 Hz,),
141.03, 129.58, 124.70, 120.22, 117.86 (d, J = 7.1 Hz), 67.28,
3.28 (t, J = 5.0 Hz), 52.43, 50.11 (d, J = 4.1 Hz), 25.90, 21.05 (d,
J = 3.3 Hz), 18.37, 13.59, -5.39. δ NMR (121 MHz, CDCl ) 2.48,
.50. HRMS (ESI): Found M+Na, 480.1949, [C21 SiPNa]
requires 480.1947.
1
2
3
4
5
6
7
8
9
s), 18.31, 13.53, -5.41. δ
(broad)). HRMS (ESI):
SiPNa] requires 522.2417.
((((E)-4-hydroxy-3-methylbut-2-en-1-yl)oxy)(phe-
P
NMR (121 MHz, CDCl
3
): 2.82 (s
522.2415,
H
3
Found M+Na,
7
5
&
[C24H42NO
6
Methyl
2
2
3
noxy)phosphoryl)-L-alaninate, 6a. The silyl ether 5a (0.12 g, 0.26
mmol, 1 eq.) was placed in a flask containing MeOH (3 mL). HCl
(1.25 M in MeOH, 0.0215 mL, 0.0268 mmol, 0.1 eq.) was then
added at 0 °C and the resulting solution was warmed to room tem-
perature while stirring for 40 min, until complete via TLC, upon
which it was neutralized with sodium bicarbonate. The mixture was
then filtered, and MeOH removed under reduced pressure. Brine
1
3
3
3
3
C
3
6
P
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
2
H
36NO
6
(10 mL) and CH
was collected and the aqueous layer was extracted with CH
mL x 3). The organic layers were combined, dried (MgSO
2
Cl
2
(10 mL) were then added. The organic layer
Cl (10
) and
2
2
Benzyl ((((E)-4-((tert-butyldimethylsilyl)oxy)-3-methylbut-2-
en-1-yl)oxy)(phenoxy)phosphoryl)-L-alaninate, 5b. Prepared
under the same procedure as described for 5a using alcohol 4 (0.16
g, 0.77 mmol, 1 eq.), benzyl phosphorchloridate 9b (0.32 g, 0.92
4
the solvents removed under reduced pressure to leave a crude
brown oil. This was then purified by column chromatography (4:1
Hex:EtOAc) to leave 6a (0.0920 g, 49%) as a colorless oil. δ
NMR (400 MHz, CDCl ): 7.32 (t, J = 7.8 Hz, 2H, 2 x CH), 7.25-
7.18 (m, 2H, 2 x CH), 7.15 (t, J = 7.3 Hz, 1H, CH), 5.67 (dtd, J =
12.5, 7.0, 1.4 Hz, 1H, CH), 4.78-4.58 (m, 2H, CH ), 4.16-3.93 (m,
3H, CH & CH), 3.71 (d, J = 8.4 Hz, 3H, CH ), 3.64-3.43 (m, 1H,
NH), 1.70 (s, 3H, CH ), 1.37 (t, J = 7.0 Hz, 3H, CH ). δ NMR
(101 MHz, CDCl ): 174.11 (2 x s), 150.78 (2 x s), 141.65 (s
H
mmol, 1.2 eq.) and dry Et
a crude yellow oil which was then purified by column chromatog-
raphy (4:1 Hex:EtOAc to 2:3 Hex:EtOAc) to afford 5b (0.1306 g,
3
N (0.22 mL, 1.5496 mmol, 2 eq.) to give
3
2
3
1%) as a colourless oil. δ
(m, 10H, Ph), 5.66 (m, 1H, CH), 5.18-5.09 (m, 2H, CH
(m, 2H, CH ), 4.13-4.04 (m, 1H, CH), 4.02 (d, J = 5.3 Hz, 2H,
CH ), 3.59-3.45 (m, 1H, NH), 1.63 (d, J = 5.1 Hz, 3H, CH ), 1.38
t, J = 6.9 Hz, 3H, CH ), 0.91 (s, 9H, CH ), 0.06 (s, 6H, 2 x CH ).
NMR (101 MHz, CDCl ): 173.32 (2 x s), 150.88 (s), 141.06 (2
H
NMR (400 MHz, CDCl
3
) δ 7.44 – 7.06
2
3
2
), 4.71-4.61
3
3
C
2
3
2
3
(broad)), 129.60, 124.76, 120.19 (t, J = 5.5 Hz), 118.48 (t, J = 7.2
Hz), 67.19, 63.49 (t, J = 5.7 Hz), 52.47 (s), 50.14 (2 x s), 20.95 (t,
(
3
3
3
δ
C
3
J = 5.3 Hz), 13.78. δ
P
NMR (121 MHz, CDCl
3
): 2.42, 2.57. HRMS
x s), 135.28, 129.59 (2 x s), 128.64 (2 x s), 128.48 (2 x s), 128.19
2 x s), 124.71 (2 x s), 120.23 (s (broad)), 117.87 (2 x s), 67.32 (2
x s (broad)), 67.18 (2 x s (broad)), 63.35 (2 x s), 50.25 (2 x s), 25.91
(2 x s), 21.11 (2 x s), 13.59 (2 x s), -5.37. δ NMR (121 MHz,
CDCl ): 2.38, 2.56. HRMS (ESI): Found M+Na, 556.2262,
PSiNa] requires 556.2260.
Isopropyl ((((E)-4-((tert-butyldimethylsilyl)oxy)-3-methylbut-
-en-1-yl)oxy)(phenoxy)phosphoryl)-L-alaninate, 5c. Prepared
(ESI): Found M+Na, 366.1081, [C15
366.1082.
H22NO PNa] requires
6
(
Benzyl
((((E)-4-hydroxy-3-methylbut-2-en-1-yl)oxy)(phe-
P
noxy)phosphoryl)-L-alaninate, 6b. Prepared following the same
procedure as described for 6a using silyl ether 5b (0.13 g, 0.24
mmol, 1 eq.) and HCl (1.25 M in MeOH, 0.19 mL, 0.24 mmol, 1
eq.) in dry MeOH (5 mL), to give a crude brown oil after 2 h. This
was then purified by column chromatography (1:1 Hex:EtOAc to
3
27 6
[C H40NO
2
under the same procedure as described for 5a using alcohol 4 (0.23
g, 1.08 mmol, 1 eq.), isopropyl phosphorchloridate 9c (0.39 g, 1.29
1:4 Hex:EtOAc) to afford 6b (0.0207 g, 20%) as a colorless oil. δ
NMR (400 MHz, CDCl ): 7.53-7.02 (m, 10H, Ph), 5.64 (dtd, J =
21.5, 7.0, 1.3 Hz, 1H, CH), 5.13 (d, J = 11.7 Hz, 2H, CH ), 4.75-
4.57 (m, 2H, CH ), 4.15-4.01 (m, 1H, CH), 3.99 (d, J = 8.9 Hz, 2H,
CH ), 3.65-3.50 (m, 1H, NH), 1.97 (s (broad), 1H, OH), 1.68 (d, J
= 4.8 Hz, 3H, CH ), 1.37 (t, J = 7.0 Hz, 3H, CH ). δ NMR (100
MHz, CDCl ): 173.45 (2 x d, J = 3.3 Hz), 150.82 (t, J = 6.1 Hz),
H
3
mmol, 1.2 eq.) and dry Et
3
N (0.30 mL, 2.16 mmol, 2 eq.) to give a
2
crude yellow oil which was then purified by column chromatog-
raphy (4:1 Hex:EtOAc) to afford 5c (0.1914 g, 36%) as a colorless
2
2
oil. δ
H
NMR (400 MHz, CDCl
3
) 7.30 (td, J = 8.5, 2.1 Hz, 2H, 2 x
3
3
C
CH), 7.20 (td, J = 7.5, 0.9 Hz, 2H, 2 x CH), 7.16-7.08 (m, 1H, CH),
3
5
2
.66 (q, J = 7.2 Hz, 1H, CH), 5.06-4.92 (m, 1H, CH), 4.75-4.59 (m,
H, CH ), 4.02 (s, 2H, CH ), 4.00-3.88 (m, 1H, CH), 3.60-3.48 (m,
), 1.38-1.32 (m, 3H, CH ), 1.26-1.17 (m,
), 0.90 (s, 9H, 3 x CH ), 0.05 (s, J = 4.2 Hz, 6H, 2 x
NMR (101 MHz, CDCl ) 173.00 (2 x d, J = 4.13 Hz),
50.92 (t, J = 6.7 Hz), 140.96, 129.57, 124.68 (s (broad)), 120.23
t, J = 4.6 Hz), 117.92 (2 x s), 69.11, 67.30, 63.28 (t, J = 5.1 Hz),
0.30 (2 x s), 25.90, 21.66 (2 x s), 21.09 (2 x s), 18.37, 13.59, -5.39.
NMR (121 MHz, CDCl ): 2.62, 2.65. HRMS (ESI): Found
M+Na, 508.2258, [C23 PSiNa] requires 508.2260.
Tert-butyl ((((E)-4-((tert-butyldimethylsilyl)oxy)-3-methylbut-
-en-1-yl)oxy)(phenoxy)phosphoryl)-L-alaninate, 5d. Prepared
141.44 (s (broad)), 135.23, 129.60, 128.65, 128.51 (2 x s), 128.20,
124.78, 120.23 (2 x d, J = 5.0 Hz), 118.81 (2 x d, J = 6.5 Hz), 67.43,
2
2
1H, NH), 1.63 (s, 3H, CH
6H, 2 x CH
CH ). δ
1
(
3
3
67.24, 63.40 (t, J = 5.8 Hz), 50.29 (2 x s), 29.70, 13.81. δ
(121 MHz, CDCl ): 2.36, 2.53. HRMS (ESI): Found M+Na,
442.1392, [C21 PNa] requires 442.1395.
P
NMR
3
3
3
3
C
3
H26NO
6
Isopropyl ((((E)-4-hydroxy-3-methylbut-2-en-1-yl)oxy)(phe-
noxy)phosphoryl)-L-alaninate, 6c. Prepared following the same
procedure as described for 6a using silyl ether 5c (0.19 g, 0.39
mmol, 1 eq.) and HCl (1.25 M in MeOH, 0.03 mL, 0.0364 mmol,
0.1 eq.) in dry MeOH (5 mL), to give a crude brown oil after 1.5 h.
This was then purified by column chromatography (1:1
5
δ
P
3
H
40NO
6
2
Hex:EtOAc) to afford 6c (0.0725 g, 49%) as a colorless oil. δ
NMR (400 MHz, CDCl ): 7.34-7.28 (m, 2H, CH), 7.21 (t, J = 7.4
Hz, 2H, 2 x CH), 7.14 (t, J = 7.3 Hz, 1H, CH), 5.73-5.61 (m, 1H,
CH), 5.08-4.91 (m, 1H, CH), 4.76-4.57 (m, 2H, CH ), 4.04 – 3.86
(m, 3H, CH & CH), 3.77-3.64 (m, 1H, NH), 2.89 (s, 1H, OH), 1.68
(s, 3H, CH ), 1.34 (t, J = 6.5 Hz, 3H, CH ), 1.22 (ddd, J = 11.7, 7.0,
5.5 Hz, 6H, 2 x CH ). δ NMR (101 MHz, CDCl ): 173.21 (2 x s),
H
under the same procedure as described for 5a using alcohol 4 (0.14
g, 0.65 mmol, 1 eq.), tert-butyl phosphorchloridate 9d (0.25 g, 0.78
mmol, 1.2 eq.) and dry NMI (0.11 mL, 1.30 mmol, 2 eq.) to give a
crude yellow oil which was then purified by column chromatog-
raphy (4:1 Hex:EtOAc) to afford 5d (0.1832 g, 56%) as a colorless
3
2
2
3
3
oil. δ
7.22 (t, J = 7.2, 2H, 2 x CH), 7.12 (t, J = 7.2 Hz, 1H, CH), 5.68 (qd,
J = 7.2, 1.3 Hz, 1H, CH), 4.74-4.62 (m, 2H, CH ), 4.02 (s, 2H,
), 3.95-3.83 (m, 1H, CH), 3.73-3.62 (m, 1H, NH), 1.64 (s, 3H,
), 1.43 (2 x s, 9H, 3 x CH ), 1.33 (2 x d, J = 3.1 Hz, 3H, CH ),
), 0.07 (s, 6H, 2 x CH ). δ NMR (101 MHz,
): 172.64 (2 x d, J = 4.1 Hz), 150.94 (t, J = 6.2 Hz), 140.76,
H
NMR (400 MHz, CDCl
3
): 7.29 (t, J = 7.2 Hz, 2H, 2 x CH),
3
C
3
150.90 (t, J = 6.2 Hz), 141.69 (s, (broad)), 129.66, 124.81, 120.28
(2 x d, J = 4.8 Hz), 118.63 (t, J = 5.7 Hz), 69.28, 67.26, 63.52 (t, J
2
CH
CH
0
2
= 5.1 Hz), 50.37 (2 x s), 21.71 (2 x s), 21.19-20.94 (m), 13.85. δ
NMR (121 MHz, CDCl ): 2.54, 2.68.
P
3
3
3
3
.91 (s, 9H, 3 x CH
3
3
C
Tert-butyl ((((E)-4-hydroxy-3-methylbut-2-en-1-yl)oxy)(phe-
noxy)phosphoryl)-L-alaninate, 6d. Prepared following the same
procedure as described for 6a using silyl ether 5d (0.1832 g, 0.3666
CDCl
3
129.49, 124.55, 120.21 (t, J = 4.8 Hz), 118.01 (2 x s (broad)), 81.69,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 7
mmol, 1 eq.) and HCl (1.25 M in MeOH, 0.0293 mL, 0.0366 mmol,
0.1 eq.) in dry MeOH (3 mL), to give a crude brown oil. This was
then purified by column chromatography (2:3 Hex:EtOAc) to af-
149.80, 129.92, 125.96, 120.57 (d, J = 5.0 Hz), 82.70 (2 x s), 51.09
(2 x s), 27.93, 20.63. δ NMR (121 MHz, CDCl ): 7.85, 8.24.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
P
3
ford 6d (0.0983 g, 74%) as a colourless oil. δ
CDCl ): 7.31 (t, J = 7.2 Hz, 2H, 2 x CH), 7.21 (dd, J = 7.6, 6.6 Hz,
H, 2 x CH), 7.13 (t, J = 7.3 Hz, 1H, CH), 5.73-5.61 (m, 1H, CH),
.75-4.59 (m, 2H, CH ), 3.99 (s (broad), 2H, CH ), 3.96-3.80 (m,
H, CH), 3.72-3.61 (m, 1H, NH), 2.96 (s (broad), 1H, OH), 1.68 (s,
), 1.43 (2 x s, 9H, CH ), 1.36-1.29 (t, J = 6.3 Hz, 3H, CH ).
NMR (101 MHz, CDCl ): 172.79 (2 x s), 150.86 (t, J = 6.1 Hz),
41.57, 129.58, 124.69, 120.22 (2 x d, J = 4.9 Hz), 118.61 (2 x s),
1.97, 67.19, 63.40 (t, J = 5.1 Hz), 50.70, 27.89, 21.11 (2 x d, J =
.6 Hz), 13.79. δ NMR (121 MHz, CDCl ): 2.70, 2.79. HRMS
PNa] requires
H
NMR (400 MHz,
ASSOCIATED CONTENT
Supporting information
The Supporting Information is available free of charge on the ACS
3
2
4
1
2
2
Publications website at DOI: xxx.
T-cell isolation, culture and activation. (PDF)
3H, CH
3
3
3
In vitro cathepsin A mediated degradation of 6d. (PDF)
δ
C
3
1
13
H NMR and C NMR spectra, HPLC data, and mass spec-
1
8
4
tra. (PDF)
Molecular formula strings. (CSV)
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
P
3
(
4
ESI): Found M+Na, 408.1554, [C18
08.1554.
H28NO
6
AUTHOR INFORMATION
Corresponding Author
*Tel: +44 (0)121 414 9533. Email: b.willcox@bham.ac.uk.
*Tel: +44 (0) 2920875821. Email: MehelouY1@cardiff.ac.uk
Author Contributions
Methyl (chloro(phenoxy)phosphoryl)-L-alaninate, 9a. Phenyl
phosphorodichloridate 7 (0.2 g, 0.95 mmol, 1 eq.) and L-alanine
methyl ester hydrogen chloride (0.13 g, 0.95 mmol, 1 eq.) were
combined in dry CH
and stirred at -78 °C for 20 minutes. Dry Et
2
CL
2
(10 mL) under an argon flow at -78 °C,
N (0.26 mL, 1.90
3
RM and RCM synthesised all of the reported compounds. MSD,
ATB and TET carried out the biological testing of the prodrugs.
CSL assisted with the NMR and mass spectroscopy studies. BEW
and YM designed and supervised the experiments. YM wrote the
manuscript and all of the authors gave approval to the final version
mmol, 2 eq.) was then added dropwise over 15 minutes and the
reaction stirred at -78 °C for a further 30 minutes. The temperature
is then raised to room temperature and the reaction stirred for a fur-
ther 3.5 hours. The solvents are removed under reduced pressure,
‡
and the mixture is filtered and washed with Et
2
O, which is removed
of the manuscript. These authors contributed equally.
under reduced pressure to give a crude oil. This is then purified by
column chromatography (7:3 Hex:EtOAc) to give 9a as a colorless
ABBREVIATIONS USED
oil (0.14 g, 54%), which is stored under argon. δ
CDCl ): 7.40-7.34 (m, 2H, 2 x CH), 7.29-7.23 (m, 3H, 2 x CH),
.66-4.49 (m, 1H, NH), 4.28-4.12 (m, 1H, CH), 3.78 (2 x s, 3H,
CH3), 1.51 (2 x dd, J = 4.1, 0.5 Hz, 3H, CH3). δ (101 MHz,
CDCl ): 173.11 (2 x d, J = 8.8 Hz), 149.73 (2 x d, J = 7.14 Hz),
29.90, 125.99, 120.54 (2 x d, J = 1.4 Hz), 52.80 (2 x s), 50.39 (2
x d, J = 1.5 Hz), 20.51 (2 x d, J = 1.5 Hz). δ NMR (121 MHz,
CDCl ): 7.62, 7.94.
H
NMR (300 MHz,
Bis-POM, bis-pivaloyloxymethyl; Bn, benzyl; HMBP, (E)-4-hy-
droxy-3-methyl-but-2-enyl monophosphate; HMBPP, (E)-4-hy-
droxy-3-methyl-but-2-enyl pyrophosphate; IPP, isopentenyl pyro-
phosphate; iPr, isopropyl; Me, methyl; NMI, N-methylimidazole;
PAg, phosphoantigen; PBMC, peripheral blood mononuclear cell;
ProPAgen: prodrug of a phosphoantigen; TBAF, tetrabutylammo-
nium fluoride; TBS: tert-butyldimethylsilyl; tBu, tert-butyl; TCR,
T-cell receptor; THF, tetrahydrofuran.
3
4
C
3
1
P
3
Benzyl (chloro(phenoxy)phosphoryl)-L-alaninate, 9b. Followed
same procedure as described for 9a using 7 (0.35 mL, 3.32 mmol,
REFERENCES
1 eq.), Et
hydrogen chloride (0.5 g, 2.32 mmol, 1 eq.) to give phos-
phorchloridate 9b (0.505 g, 95%). δ NMR (300 MHz, CDCl):
.42-7.32 (m, 7H, Ph), 7.25-7.20 (m, 3H, Ph), 5.21 (2 x s, 2H,
CH2), 4.34-4.15 (m, 2H, CH & N-H), 1.52 (dd, J = 6.6, 2.4 Hz, 3H,
CH3). δ NMR (101 MHz, CDCl ): 172.52 (dd, J = 11.6, 8.7 Hz),
49.75 (dd, J = 11.6. 8.7 Hz), 135.05 (d, J = 5.8 Hz, C-10), 129.92,
128.68, 128.59, 128.33, 125.99, 120.57, 67.60, 50.67, 20.47 (t, J =
4.5 Hz). δ NMR (121 MHz, CDCl): 7.49, 7.85.
3
N (0.63 mL, 4.64 mmol, 2 eq.) and L-alanine benzyl ester
1.
Dimova, T.; Brouwer, M.; Gosselin, F.; Tassignon, J.; Leo,
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H
3
7
2.
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C
3
1
P
3
Isopropyl (chloro(phenoxy)phosphoryl)-L-alaninate, 9c. Fol-
lowed same procedure as described for 9a using 7 (0.62 mL, 4.18
3. Shen, Y.; Zhou, D.; Qiu, L.; Lai, X.; Simon, M.; Shen, L.;
Kou, Z.; Wang, Q.; Jiang, L.; Estep, J.; Hunt, R.; Clagett, M.;
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Gschwind, R. M.; Kollas, A. K.; Beck, E.; Wiesner, J.; Eberl,
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mmol, 1 eqvs), Et
isopropyl ester hydrogen chloride (0.700 g, 4.18 mmol, 1 eqvs) to
give phosphorchloridate 9c (1.185 g, 93%). δ NMR (300 MHz,
CDCl ): 7.41- 7.34 (m, 2H, 2 x CH), 7.30- 7.21 (m, 3H, 3 x CH),
5.15-5.01 (m, 1H, CH), 4.50-4.31 (m, 1H, NH), 4.22-4.04 (m, 1H,
CH), 1.50 (dd, J = 7.0, 2.1 Hz, 3H, CH), 1.34-1.22 (m, 6H, 2 x
CH ). δ NMR (101 MHz, CDCl ): 172.15 (dd, J = 13.9, 9.2 Hz),
49.76 (t, J = 7.0 Hz), 129.93, 125.98, 120.56 (d, J = 4.9 Hz), 69.79
d, J = 10.5 Hz), 50.70 (2 x s), 21.67 (2 x s), 20.55 (d, J = 4.4 Hz).
NMR (121 MHz, CDCl ): 7.76, 8.11.
3
N (1.13 mL, 8.36 mmol, 2 eqvs) and L-alanine
H
3
3
3
C
3
1
(
δ
P
3
tert-butyl (chloro(phenoxy)phosphoryl)-L-alaninate, 9d. Fol-
lowed same procedure as described for 9a using 7 (0.22 mL, 1.5
mmol, 1 eq.), Et
butyl ester hydrogen chloride (0.27 g, 1.5 mmol, 1 eq.) to give
phosphorchloridate 9d (0.44 g, 90%). δ NMR (400 MHz, CDCl):
.37 (t, J = 7.8 Hz, 2H, 2 x CH), 7.30-7.22 (m, 3H, 2 x CH), 4.46-
.24 (m, 1H, NH), 4.16-3.97 (m, 1H, CH), 1.50-1.46 (m, 12H, 4 x
). δ NMR (101 MHz, CDCl): 171.77 (dd, J = 15.8, 9.4 Hz),
3
N (0.42 mL, 3 mmol, 2 eq.) and L-alanine tert-
H
3
7
4
CH
3
C
3
155-158.
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Journal of Medicinal Chemistry
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Specific activators of Vγ9/Vδ2 T-cells, EC50 = 0.45 to 11 nM
Potent lysis of bladder carcinoma cancer cells
O
R
O P O
HN
O
O
O R1
1
One ProPAgen > 1000-times more potent than Zoledronate
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