Development of novel PP2A activators for use in the treatment of acute myeloid leukaemia

Article abstract of DOI:10.1039/c6ob00556j

AAL(S), the chiral deoxy analog of the FDA approved drug FTY720, has been shown to inhibit proliferation and apoptosis in several cancer cell lines. It has been suggested that it does this by activating protein phosphatase 2A (PP2A). Here we report the synthesis of new cytotoxic analogs of AAL(S) and the evaluation of their cytotoxicity in two myeloid cell lines, one of which is sensitive to PP2A activation. We show that these analogs activate PP2A in these cells supporting the suggested mechanism for their cytotoxic properties. Our findings identify key structural motifs required for anti-cancer effects.

Full text of DOI:10.1039/c6ob00556j

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Development of novel PP2A activators for use in
the treatment of acute myeloid leukaemia†
Cite this: DOI: 10.1039/c6ob00556j
a
b,c
d
b,c
Hamish D. Toop, Matthew D. Dun, Bryony K. Ross, Hayley M. Flanagan,
b,c
a
Nicole M. Verrills and Jonathan C. Morris*
AAL(S), the chiral deoxy analog of the FDA approved drug FTY720, has been shown to inhibit proliferation
and apoptosis in several cancer cell lines. It has been suggested that it does this by activating protein
phosphatase 2A (PP2A). Here we report the synthesis of new cytotoxic analogs of AAL(S) and the evalu-
ation of their cytotoxicity in two myeloid cell lines, one of which is sensitive to PP2A activation. We show
that these analogs activate PP2A in these cells supporting the suggested mechanism for their cytotoxic
properties. Our findings identify key structural motifs required for anti-cancer effects.
Received 14th March 2016,
Accepted 18th April 2016
DOI: 10.1039/c6ob00556j
www.rsc.org/obc
Introduction
FTY720 (1) (fingolimod, Gilenya) is approved by the FDA as an
1
immunosuppressant to treat multiple sclerosis. The immuno-
suppressive properties of this molecule arise from in vivo phos-
phorylation of one of the alcohol groups, to afford the active
2
metabolite, FTY720-P (2) (Fig. 1). Interestingly, non-phosphory-
lated FTY720 (1) has been shown to induce apoptosis in a
3
number of cancer cell lines. While the exact mechanism of
action is not known, apoptosis is achieved by indirectly activat-
ing protein phosphatase 2A (PP2A), which is an important phos-
4
phatase involved in growth factor signalling. Inhibition of PP2A
has been found to be crucial for the oncogenic effects of tyro-
5
sine kinases such as, BCR/ABL, c-KIT and FLT3-ITD and has
been shown to down regulate plasma membrane transporters of
6
amino acids and glucose. Importantly, FTY720 (1) is able to
eliminate cancer cells which are resistant to current therapeutics
7
without disruption to normal blood or bone marrow cells.
Fig. 1 Structures of FTY720 (1) and AAL(S) (3) and the regions of AAL(S)
we chose to target as part of a structure–activity relationship study.
Our lab has shown that tumorigenic, factor-independent
FDC.P1 myeloid cells transduced with the mutant c-KIT onco-
gene (D816V), are more sensitive to PP2A activation than cells
expressing wildtype c-KIT, or those transduced with an empty limits FTY720’s utility as a cancer drug. In contrast, AAL(S) (3),
vector (EV), and that FTY720 (1) inhibits proliferation of the the chiral deoxy analog of FTY720 (1), has been shown to
D816V cell line with 1.8 times selectivity, when compared to retain the PP2A activation and cytotoxic properties of FTY720
8
the EV cells. However, the in situ phosphorylation of FTY720 (1), but is not phosphorylated in vivo and as such, does not
8
have the same immunosuppressive properties as FTY720 (1).
As a consequence, AAL(S) (3) has emerged as a molecule that
a
could have value in the development of new cancer thera-
peutics. With this information, we sought to develop the cyto-
toxic properties of AAL(S) (3).
Recent work in our laboratories has focused on the develop-
ment of an efficient synthetic strategy to access AAL(S) analogs
and in a preliminary study we found that modifications to the
hydrophobic tail moiety of AAL(S) (3) can have a major impact
School of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia.
E-mail: jonathan.morris@unsw.edu.au
b
School of Biomedical Sciences and Pharmacy, The University of Newcastle,
Callaghan, NSW 2308, Australia
c
Hunter Medical Research Institute, Newcastle, NSW 2305, Australia
d
Calvary Mater Hospital, Newcastle, NSW 2298, Australia
Electronic supplementary information (ESI) available: Synthetic procedures
†
and characterisation data for all new compounds. See DOI: 10.1039/c6ob00556j
This journal is © The Royal Society of Chemistry 2016
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9
9
on the cytotoxicity in K562 leukaemia cells. This paper length of the chain can have profound effects on cytotoxicity.
extends this study and provides further insights into the role Testing them in the FDC.P1-c-KIT-D816V cell line confirmed
of each of the substituents of the quaternary aminoalcohol this trend. While activity is completely lost when the tail is
head group and the phenoxy ether group (Fig. 1) in an acute shortened (<4 carbons long), is a benzyl group 10, or a hydro-
myeloid leukaemia model. A select number of the compounds philic PEG-like tail 11, the OC8 analog 6 was found to have a
were also tested for their ability to activate PP2A in cells to similar activity against D816V cells. However, there was
support the proposed mechanism of action.
deterioration of the selectivity for the D816V cells over the EV
cells. Consequently, the OC7 tail was retained throughout the
rest of our study.
Results and discussion
Role of the 1-alcohol group (X)
As a starting point in this investigation, we examined the As shown in Scheme 1, three analogs 12–14 (where X = H,
ability of AAL(S) (3) to inhibit the growth of D816V and EV F and OMe) were synthesised to investigate whether the
cells (Table 1). AAL(S) (3) behaves similarly to FTY720 (1), indu- 1-alcohol group had a role as either a hydrogen bond donor or
cing cytotoxicity in D816V cells with an IC50 of 3.6 μM and acceptor, or whether it was required at all.
7
.0 μM for the EV cells. This results in a slightly improved
Both the 1-methoxy analog 12 and the 1-fluoro analog 13
selectivity index of 1.9 compared to FTY720 (1). This confirms could be generated from AAL(S) (3). The 1-methoxy analog 12
9
that the AAL(S) scaffold is an interesting starting point for was synthesised in a 4 step sequence, as previously reported.
developing FTY720 analogs that could be used in the treat- The synthesis of the 1-fluoro analog 13 began by firstly Boc-
ment of leukaemia.
2
protection of the amino group of AAL(S) (3), using Boc O and
The hydrophobic tail analogs 4–11 have been previously saturated aqueous NaHCO3 solution in EtOAc at reflux, to
synthesised by our group and our preliminary cytotoxicity afford N-Boc AAL(S) in 83% yield. This material was converted
results, using the K562 leukaemia cell line, indicated that the into a cyclic sulfamidate, following the protocol developed by
+
Table 1 Cytotoxicity data for analogs of AAL(S) in FDC.P1 myeloid cells WT (EV) and mutant c-KIT (D816V) cells and, for selected analogs, in vivo
PP2A activation data
a
IC50 (μM)
b
Compound number
R
C
OC
OMe
OC
OC
OC
OC10
OC12
OBn
X
Y
Z
EV
D816V
Selectivity
PP2A activation (%)
c
FTY720 (1)
AAL(S) (3)
8
H
17
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
F
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
2
2
2
2
2
2
2
2
2
2
2
2
2
CH
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
2
OH
6.3
7.0
>9
3.6
3.7
>9
1.8c
150.1 ± 5.1
139.5 ± 6.7
7
H
15
1.9
0
d
4
5
6
7
8
9
ND
4
H
8
H
9
H
9
>9
>9
0
1.4
ND
c
17
5.0
4.2
>9
>9
>9
3.6
4.0
>9
>9
>9
129.7 ± 1.5
115.1 ± 6.4
ND
ND
ND
ND
136.8 ± 6.3
ND
127.5 ± 11.6
118.5 ± 9.1
ND
ND
ND
ND
ND
ND
131.7 ± 1.9
137.0 ± 1.3
19
H
H
1.1
0
0
21
25
10
11
12
13
14
16
17
18
19
20
22
23
24
25
0
O(CH
2
CH
2 2
O) Me
>9
>9
0
1.4
c
OC
OC
OC
OC
OC
OC
OC
OC
OC
OC
OC
7
H
7
H
7
H
7
H
7
H
7
H
7
H
7
H
7
H
7
H
7
H
15
15
15
15
15
15
15
15
15
15
15
6.1
6.9
4.0
>9
>9
>9
5.8
7.0
>9
7.6
8.4
4.1
4.3
6.7
3.1
7.5
>9
1.0
1.3
≥1.2
0
H
OH
OH
OH
OH
OH
NMe
2
NHAc
OH
NH
NH
>9
0
2
5.9
7.1
>9
6.6
5.7
2.7
1.0
1.0
0
1.2c
1.5c
1.5
2
Et
–O(CO)NH–
Me
Me
CH
Me
NH
OH
OH
2
OH
NH
NH
2
2
2
OH
8 17
C H
a
5c b
Cells were treated with increasing concentrations of compound for 48 h. and viability determined using the resazurin assay.
816 V myeloid
cells were treated with 2.5 μM of compound for 12 h. PP2A activity was measured in PP2A immunoprecipitates by quantitation of phosphate
5
c
c
release from a PP2A-specific phospho-peptide. Control was determined in untreated D816V cells as 100%. Data is mean ± SEM, n = 3. p < 0.01
d
compared with EV, Student’s t test. ND: not determined.
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Scheme 1 Reagents and yields (a) n-BuLi, THF, −78 °C, then MeI, −78 °C → 0 °C, 96%; (b) CsF, H15
C
7
Br, DMF, rt, 72–88%; (c) TFA, H
NSO , 50% v/v 2 M aq. NaOH/THF, rt; (g) 2 M
, py, MeCN, −40 °C → −10 °C, diastereomer 1 48%: diastereomer 2 46%; (i) RuCl , NaIO , 3 : 1 MeCN/H O,
NF, MeCN, Δ, then 6 M aq HCl, Δ, 88%; (k) NaCNBH , (CH O) , AcOH, MeCN, 0 °C → rt, 54%; (l) AcCl, NEt , CH Cl , 0 °C → rt,
5%; (m) n-BuLi, THF, −78 °C, then MeOH, −78 °C, then 0 °C, 27% (59% 21); (n) n-BuLi, THF, −78 °C, then EtI, −78 °C → 0 °C, 87%; (o) KOH, TsCl,
2
O, MeCN, rt,
2
5–99%; (d) LiAlH
aq. HCl, MeCN, Δ, 82% (2 steps); (h) SOCl
°C, 85%; ( j) n-Bu
4
, THF, 0 °C → rt, 44–86%; (e) Boc
2
O, sat. aq. NaHCO
3
, EtOAc, Δ, 83%; (f) MeI, n-Bu
4
4
2
3
4
2
0
4
3
2
n
3
2
2
3
Et
0
Et
2
O, Δ, 90%; (p) KCN, H
°C → rt, 83%; (s) TMSEOCONH
2
O, MeOH, rt, 45%.
2
SO
4
, AcOH, 0 °C → rt, 97%; (q) 6 M aq. HCl, Δ, 96%; (r) K
2
OsO
4
·2H
2
O, K
2
CO
3
, K
3
Fe(CN)
6
, (DHQD)
2
PHAL, 1 : 1 t-BuOH/H
2
O,
2
, (DHQD) AQN, K OsO ·2H O, 1,3-dichloro-5,5-dimethylhydantoin, NaOH, 1 : 1 n-PrOH/H
2
2
4
2
2
O, 77%; (t) 2 M HCl in
1
0
Hinterding and co-workers for AAL(R), by treatment of Boc- examine its role by either functionalising the amino group or
AAL(S) with SOCl and py in MeCN. This resulted in a mixture by looking at a isosteric replacement. The two amino analogs
2
of diastereomeric cyclic sulfimidates which were oxidised to (2-N,N-dimethylamine 16 and 2-N-acetyl 17) have been pre-
the cyclic sulfamidate using RuCl and NaIO in 3 : 1 MeCN/ viously synthesised, but their biological activity against leukae-
3
4
9
H
2
O in 80% yield over the two steps. Ring opening of this mia cell lines has not been examined.
intermediate, using TBAF in MeCN at reflux, followed by The isosteric oxygen analog 18 was synthesised, starting
in situ deprotection of the Boc group with 6 M aqueous HCl from the alkene 15 that was required in the synthesis of 14,
1
3
solution at reflux resulted in the formation of 1-fluoro analog using a Sharpless dihydroxylation protocol (K
3 in 88% yield. CO , K Fe(CN) and (DHQD) PHAL in 1 : 1 t-BuOH/H
The achiral amine 14, was generated from the readily avail- 0 °C → rt) to afford dihydroxy analog 18 in 83% yield.
2
OsO
4
·2H
2
O,
1
K
2
3
3
6
2
2
O at
1
1
able alkene 15 by using the Ritter protocol (KCN in H
2
SO
4
,
Significantly, all the analogs varying the 2-amino group lost
AcOH, 0 °C → rt followed by treatment with 6 M aqueous HCl cytotoxicity in both the D816V and EV cell lines, suggesting
1
2
solution at reflux) to provide 14 in 94% yield over two steps.
that the amine could be involved in an electrostatic interaction
As detailed in Table 1, the 1-fluorinated analog 13 lost and that the binding pocket must be small and cannot tolerate
activity and selectivity, while the 1-methoxy 12 analog lost any steric bulk.
some potency but retained the selectivity. This data provides
some evidence that the 1-hydroxy group of the AAL(S) scaffold Significance of the 2-methyl group (Z)
could be acting as a hydrogen bond acceptor. In contrast, the
The role of the 2-methyl group was investigated by preparing a
1
-des-hydroxy analog 14 displayed a significant improvement
2
-des-methyl analog 19 and 2-ethyl analog 20. The 2-ethyl
analog 20 was synthesised by treating 21 with n-BuLi in THF at
78 °C and quenching the resulting anion with ethyl iodide to
in cytotoxicity relative to AAL(S) (3) and FTY720 against the
D816V cells, albeit, with a decrease in selectivity (1.3) when
compared to the EV cells.
−
14
afford the corresponding bis-lactim ether in 87% yield. The
synthesis was completed using our two-step one-pot de-
protection/alkylation protocol to install the hydrophobic tail,
Role of the amino group (Y)
The amino group of AAL(S) (3) would be expected to be proto- hydrolysis and reduction of the Schöllkopf group in 13% yield
nated at physiological pH and thus, may be involved in an over the three steps. In line with results reported by Hinterd-
1
5
electrostatic interaction with a protein target. It was decided to ing, hydrolysis of the Schöllkopf group for this substrate
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proved troublesome due to increased bulk of the ethyl group CONH
2
,
(DHQD)
2
AQN,
K
2
OsO
4
·2H
2
O,
1,3-dichloro-5,5-
around the quaternary stereocentre resulting in the isolation dimethyl-hydantoin and NaOH in 1 : 1 n-PrOH/H O) followed
2
of a considerable amount of partially hydrolysed products. by TMSE deprotection with 2 M ethereal HCl in MeOH to
The 2-des-methyl analog 19 was synthesised by treating bis- afford 23 in 35% yield over 2 steps.
lactim ether 21 with n-BuLi in THF at −78 °C. The resulting
The cyclic oxazolidinone 22 and AAL(S) regioisomer 23
anion was quenched with MeOH and quickly warmed to 0 °C analogs also displayed poor activity in both D816V and the EV
to epimerise the stereocentre. This resulted in the formation cell lines, which strengthens our hypothesis that the 2-amino
of the desired material 19 in 27% yield and recovery of the group is involved in an electrostatic interaction in the binding
starting material in 59% yield. While 19 should be accessed site.
much more efficiently by using the alternative enantiomer of
Schöllkopf’s reagent this epimerisation protocol allowed ready Phenoxy group
access to complete the synthesis of the analog.
As the FTY720 tail is a C8 hydrocarbon, two FTY720-AAL(S)
Thus, the synthesis of 2-des-methyl analog 21 was com-
hybrid molecules 24 and 25 were synthesised so we could
pleted in 3 steps using the same two step one-pot de-
examine the importance of the phenoxy ether group of AAL(S)
protection/alkylation protocol to afford 21 in 31% overall yield.
These analogs were found to be less cytotoxic, and with no
selectivity for the D816V cells, which suggests that the methyl
group is the optimal size and/or is helping maintain a favour-
able conformation of the aminoalcohol head group (Table 1).
8b,17
(
3) (Scheme 2). The previously reported
O-FTY720 analog
24 was prepared starting from TBS-protected ether 26 and
using our two-step one-pot deprotection/alkylation strategy to
install the tail. The synthesis was completed in 58% yield over
the three steps.
To synthesise carbo-AAL(S) 25, the counterpart to O-FTY720
Other aminoalcohol head group analogs
(
24), it was decided to utilise the previously prepared phenol
9
To synthesise the 1-alcohol (X) analogs it was initially envis- 27. Conversion of 27 to the triflate 28 was achieved using Tf O
2
aged that the Boc-protected AAL(S) intermediate could be and pyridine in CH Cl in 46% yield. Despite many attempts
2
2
transformed into a cyclic aziridine which, when treated with at conducting a Sonagashira reaction on this substrate with
nucleophiles, could ring-open to substitute this position. 1-octyne, none of the desired material could be generated. To
However, treating Boc-AAL(S) under standard conditions used overcome this, (S)-Schöllkopf reagent 29 was alkylated with
11
2
to generate aziridines (KOH and TsCl in refluxing Et O) readily available 1-(2-iodoethyl)-4-octylbenzene (30), followed
resulted only in the isolation of oxazolidinone 22 in 90% yield, by reaction with methyl iodide to generate bis-lactim ether 31
which must arise through intramolecular attack of the hydroxyl in 43% yield over the two steps. The synthesis was completed
group.
by hydrolysis and reduction of the Schöllkopf group to afford
The AAL(S) regioisomer analog 23 was synthesised from carbo-AAL(S) 25 in 45% yield over the two steps, as shown in
1
6
alkene 15 using an aminohydroxylation reaction (TMSEO- Scheme 2.
Scheme 2 Reagents and yields (a) CsF, H15
°C, 46%; (e) n-BuLi, THF, −78 °C, then 30, −78 °C → 0 °C, 83%; (f) n-BuLi, THF, −78 °C, then MeI, −78 °C → 0 °C, 52%; (g) TFA, H
h) LiAlH , THF, 0 °C → rt, 69%.
C
7
Br, DMF, rt, 94%; (b) LiAlH
4
, THF, 0 °C → rt; (c) conc. HCl, EtOH, Δ, 62% (2 steps); (d) Tf
2 2 2
O, py, CH Cl
0
(
2
O, MeCN, rt, 65%;
4
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alcohol head group of AAL(S) (3) is superior to that of FTY720
(
1) in the design of PP2A activators. The structure–activity
relationship data generated here will be valuable in our efforts
to elucidate how these PP2A activators induce cell death in
cancer cell lines.
Fig. 2 SAR conclusions made from our study on the AAL(S) scaffold.
Experimental
Chemistry
1
1
Synthesis of (2S)-1-fluoro-4-(4′-heptyloxyphenyl)-2-methyl-
butan-2-amine (13). (a) Thionyl chloride (0.12 mL, 1.67 mmol)
and pyridine (0.27 mL, 3.34 mmol) were added successively,
dropwise to a solution of (2S)-t-butyl(1-hydroxy-4-(4′-heptyloxy-
The biological data for these two analogs were found to be
quite revealing, with O-FTY720 (24) having a much higher IC50
than AAL(S) (3) and FTY720 (1). In contrast, the carbo-AAL(S)
analog 25 showed significantly improved cytotoxicity in com-
parison to both AAL(S) (3) and FTY720 (1), but importantly,
maintained excellent selectivity for the D816V cells over the EV
cells.
9
phenyl)-2-methylbutan-2-yl)carbamate (0.26 g, 0.67 mmol) in
acetonitrile (15 mL) at −40 °C (dry ice/acetonitrile). The solu-
tion was allowed to warm slowly, in the cold bath, to −10 °C
over 2.5 h. The reaction mixture was poured onto 1 M aqueous
hydrochloric acid and extracted with ethyl acetate (×3). The
organic extracts were combined and washed with saturate
aqueous sodium bicarbonate, water and brine, the dried
PP2A activation data
To further justify that the analogs that we had prepared were
cytotoxic in our acute myeloid leukaemia model by activating
PP2A, a select number of compounds were tested by treating
D816V cells with 2.5 μM drug for 12 hours then immuno-
purifying PP2A complexes and measuring their phosphatase
(
2 4
Na SO ). The solvent was removed under reduced pressure to
afford (4S)-t-butyl-4-(4′-heptyloxyphenylethyl)-4-methyl-1,2,3-
oxathiazolidine-3-carboxylate-2-oxide as a 1 : 1 mixture of dia-
stereomers which were purified by flash chromatography on
silica gel, eluting with 5% ethyl acetate/n-hexane, to afford one
of the diastereomers as a clear colourless oil (0.14 g, 48%).
5
c
activity against a PP2A-specific phospho-peptide. Pleasingly,
all of the compounds that we tested showed increased PP2A
activity relative to the control, untreated cells (Table 1). Of par-
ticular interest was the result for the 1-des-hydroxy analog 14
as it had been hypothesised that the potent cytotoxicity may be
a result of a mechanism not involving PP2A as the simplified
structure is reminiscent of a detergent. However, this analog
also showed increased PP2A activity, which supported our
belief that these compounds operate by activating PP2A.
2
7.2
1
[α]
D
= −36 (0.5, CHCl
3 3
); H NMR (300 MHz; CDCl ) δ 0.89 (t,
J = 6.8 Hz, 3H), 1.26–1.47 (m, 8H), 1.45 (s, 3H), 1.54 (s, 9H),
1
2
4
.71–1.81 (m, 2H), 2.00–2.10 (m, 1H), 2.28–2.39 (m, 1H),
.44–2.55 (m, 1H), 2.59–2.69 (m, 1H), 3.92 (t, J = 6.6 Hz, 2H),
.28 (d, J = 8.9 Hz, 1H), 4.96 (br d, J = 8.9 Hz, 1H), 6.79–6.84
1
3
(
m, 2H), 7.04–7.10 (m, 2H); C NMR (75 MHz; CDCl
3
) δ 14.2,
2
1
2.7, 26.2, 28.4, 29.2, 29.5, 30.2, 31.9, 40.0, 68.2, 83.8, 114.7,
−
1
29.3, 133.0, 157.7; IR (NaCl, neat) 1722 cm ; HRMS (ESI-MS):
+
m/z calcd for C23
5
H37NO SNa [M + Na] 462.2290, found
4
62.2277. Further elution afforded the other diastereomer as a
Conclusions
2
7.2
1
clear, colourless oil (0.15 g, 51%). [α]
= +40 (0.5, CHCl ); H
3
D
3
The synthesis of eight novel analogs of AAL(S) were developed NMR (300 MHz; CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), 1.26–1.47 (m,
so that an SAR investigation of the role of each of the substitu- 8H), 1.53 (s, 9H), 1.64 (s, 3H), 1.72–1.81 (m, 2H), 1.87 (td, J =
ents of the aminoalcohol head group as well as the phenoxy 12.8, 5.2 Hz, 1H), 2.21 (td, J = 12.8, 4.6 Hz, 1H), 2.41 (td, J =
ether group in the hydrophobic tail of AAL(S) (3) could be 12.8, 4.8 Hz, 1H), 2.58 (td, J = 12.8, 5.2 Hz, 1H), 3.92 (t, J = 6.6
carried out. These analogs, as well as fourteen previously Hz, 2H), 4.59 (d, J = 9.2 Hz, 1H), 4.84 (br d, J = 9.2 Hz, 1H),
1
3
reported analogs, were evaluated as cytotoxic agents using an 6.79–6.84 (m, 2H), 7.03–7.08 (m, 2H); C NMR (75 MHz;
acute myeloid leukaemia model which is sensitive to PP2A CDCl ) δ 14.2, 22.7, 26.2, 28.4, 29.2, 29.4, 30.3, 31.9, 68.2, 83.9,
3
−1
activation. The analogs showed varying cytotoxicity and 114.7, 129.2, 132.9, 157.7; IR (NaCl, neat) 1724 cm ; HRMS
revealed that some key structural motifs are critical to the cyto- (ESI-MS): m/z calcd for C H NO SNa [M + Na] 462.2290,
+
2
3
37
5
toxicity of the AAL(S) scaffold (Fig. 2). Importantly, it was found 462.2279.
shown that these analogs activate PP2A in oncogenic mutant (b) Ruthenium(III) chloride (17 mg, 65 μmol) and sodium
c-KIT-D816V myeloid cells confirming the suggested mechan- periodate (0.21 g, 0.99 mmol) were added successively to a
ism of action for these compounds. solution of (4S)-t-butyl-4-(4′-heptyloxyphenylethyl)-4-methyl-
+
In particular, carbo-AAL(S) 25 had increased cytotoxicity in 1,2,3-oxathiazolidine-3-carboxylate-2-oxide (0.29 g, 0.66 mmol,
the D816V cell line albeit, with a small drop in selectivity over 50 : 50 mixture of diastereomers) in 3 : 1 acetonitrile : water
the non-tumorigenic control myeloid cell line. The cytotoxicity (40 mL) at 0 °C. The solution was stirred at 0 °C for 1 h then,
data obtained for O-FT720 (24) and carbo-AAL(S), and in com- the reaction solution was diluted with water and extracted with
parison to AAL(S) (3) and FTY720 (1), suggests that the amino- diethyl ether (×3). The organic extracts were combined and
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washed with saturated aqueous sodium bicarbonate, water purified by flash chromatography on silica gel, eluting with
and brine, then dried (Na SO ). The solvent was removed 10% ethyl acetate/n-hexane, to afford 4-(4′-(Heptyloxy)phenyl)
2
4
1
under reduced pressure and the crud material purified by butane-2-one as a clear colourless oil (1.94 g, 91%). H NMR
flash chromatography on silica gel, eluting with 10% ethyl (300 MHz; CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), 1.31–1.49 (m, 8H),
3
acetate/n-hexane, to afford (4S)-t-butyl-4-(4′-heptyloxyphenyl- 1.72–1.81 (m, 2H), 2.12 (s, 3H), 2.69–2.74 (m, 2H), 2.81–2.86
ethyl)-4-methyl-1,2,3-oxathiazolidine-3-carboxylate-2,2-dioxide (m, 2H), 3.92 (t, J = 6.6 Hz, 2H), 6.79–6.83 (m, 2H), 7.06–7.10
2
7.2
13
as a clear colourless oil (0. 26 g, 85%). [α]
D
= +22 (0.5, (m, 2H); C NMR (75 MHz; CDCl
3
) δ 14.2, 22.7, 26.1, 29.0,
1
CHCl ); H NMR (300 MHz; CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), 29.2, 29.4, 30.2, 31.9, 45.6, 68.1, 114.6, 129.3, 132.9, 157.7,
.26–1.47 (m, 8H), 1.57 (s, 9H), 1.61 (s, 3H), 1.72–1.81 (m, 2H), 208.3; IR (NaCl, neat) 1717 cm ; HRMS (ESI-MS): m/z calcd
3
3
−
1
1
1
6
6
+
.96 (m, 1H), 2.33–2.44 (m, 1H), 2.49–2.68 (m, 2H), 3.92 (t, J = for C17
.6 Hz, 2H), 4.16 (d, J = 9.2 Hz, 1H), 4.41 (d, J = 9.2 Hz, 1H), (b) A solution of n-butyllithium in hexanes (4.00 mL, 1.4 M,
.80–6.85 (m, 2H), 7.04–7.09 (m, 2H); C NMR (75 MHz; 5.40 mmol) was added dropwise to a solution of methyl-
) δ 14.2, 22.7, 23.4, 26.2, 28.1, 29.2, 29.37, 29.43, 31.9, triphenylphosphonium bromide (2.06 g, 5.76 mmol) in freshly
8.7, 65.0, 68.2, 73.9, 85.4, 114.8, 129.3, 132.2, 148.4, 157.9; IR distilled THF (50 mL) at room temperature. The solution was
2
H26NO Na [M + Na] 285.1831, found 285.1827.
1
3
CDCl
3
3
(
C
−
1
NaCl, neat) 1734 cm ; HRMS (ESI-MS): m/z calcd for stirred at room temperature for 15 min where it had turned
+
23
H
37NO
6
SNa [M + Na] 478.2239, found 478.2228.
c) A solution of tetra-n-butylammonium fluoride in THF one (1.01 g, 3.84 mmol) in freshly distilled THF (15 mL) was
0.28 mL, 1.0 M, 0.28 mmol) was added dropwise to a solution added dropwise after which, the reaction solution was stirred
of (4S)-t-butyl-4-(4′-heptyloxyphenylethyl)-4-methyl-1,2,3- at room temperature for an additional 5 h. The reaction was
dark orange. A solution of 4-(4′-(Heptyloxy)phenyl)butane-2-
(
(
oxathiazolidine-3-carboxylate-2,2-dioxide (51 mg, 0.11 mmol) quenched with water and the mixture extracted with n-pentane
in acetonitrile (3 mL) at room temperature. The solution was (×3). The organic extracts were combined and washed with
heated at reflux for 3 h. then, the solution was allowed to cool water and brine, then dried (Na SO ). The solvent was removed
2 4
to room temperature and 6 M aqueous hydrochloric acid solu- under reduced pressure and the crude material purified by
tion was added dropwise. The reaction mixture was heated at flash chromatography on silica gel, eluting with n-hexane, to
reflux for a further 3 h. after which, it was allowed to cool to afford the title compound 15 as a clear colourless oil (0.83 g,
1
room temperature. The acetonitrile was removed under 83%). H NMR (300 MHz; CDCl ) δ 0.90 (t, J = 6.8 Hz, 3H),
3
reduced pressure. The residue was neutralised with solid 1.26–1.50 (m, 8H), 1.72–1.82 (m, 2H), 1.77 (s, 3H), 2.26–2.31
sodium bicarbonate, diluted with water and extracted with (m, 2H), 2.67–2.72 (m, 2H), 3.93 (t, J = 6.6 Hz, 2H), 4.70–4.71
ethyl acetate (×3). The organic extracts were combined and (m, 1H), 4.73–4.74 (m, 1H), 6.79–6.84 (m, 2H), 7.07–7.12 (m,
1
3
washed with water and brine, then dried (Na
was removed under reduced pressure and the crude material 31.9, 33.5, 40.0, 68.2, 110.3, 114.5, 129.3, 134.2, 145.7, 157.4.
purified by flash chromatography on silica gel, eluting with 4-(4′-Heptyloxyphenyl)-2-methylbutan-2-amine (14).
% triethylamine/ethyl acetate, to afford the title compound Under a static atmosphere, a solution of concentrated sulfuric
2 4 3
SO ). The solvent 2H); C NMR (75 MHz; CDCl ) δ 14.2, 22.8, 26.2, 29.2, 29.5,
(a)
1
1
2
6.2
3 as a clear colourless oil (29 mg, 88%) [α]
D
= +4 (0.5, acid (0.2 mL, 1.11 mmol) in acetic acid (1 mL) was added drop-
1
CHCl ); H NMR (600 MHz; CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), wise to a solution of 1-(heptyloxy)-4-(3′-methylbut-3′-enyl)
3
3
1
1
1
2
2
1
.14 (d, JH–F = 1.9 Hz, 3H), 1.28–1.47 (m, 10H), 1.67–1.73 (m, benzene (0.19 g, 0.73 mmol) and potassium cyanide (48 mg,
H), 1.74–1.79 (m, 2H), 2.56–2.66 (m, 2H), 3.92 (t, J = 6.6 Hz, 0.74 mmol) in acetic acid (1 mL) at 0 °C. The solution was
H), 4.12 (dd, J = 17.7, 8.8 Hz, 1H), 4.20 (dd, J = 17.7, 8.8 Hz, allowed to slowly warm to room temperature, in the cold bath,
H), 6.81–6.83 (m, 2H), 7.08–7.10 (m, 2H); C NMR (150 MHz; over 24 h. The reaction solution was basified to pH 12 with
) δ 14.2, 22.7, 23.8 (d, JC–F = 4.2 Hz), 26.1, 29.2, 29.3, 2 M aqueous sodium hydroxide solution and extracted with
9.4, 31.9, 41.2 (d, JC–F = 2.9 Hz), 52.2 (d, JC–F = 17.5 Hz), 68.2, diethyl ether (×3). The organic extracts were combined and
0.9 (d, JC–F = 173.3 Hz), 114.6, 129.2, 134.1, 157.5; IR (NaCl, dried (Na SO ). The solvent was removed under reduced
1
3
CDCl
2
9
3
2
4
−
1
neat) 3294, 3370 cm ; HRMS (ESI-MS): m/z calcd for pressure and the crude material purified by flash chromato-
C H FNO [M + H] 296.2390, found 296.2384.
+
graphy on silica gel, eluting with 50% ethyl acetate/n-hexane,
1
8
31
Synthesis of 1-(heptyloxy)-4-(3′-methylbut-3′-enyl)benzene to afford (2S)-N-(4-(4-heptyloxyphenyl)-1-hydroxy-2-methyl-
(
15). (a) 4-(4-Hydroxyphenyl)butan-2-one (1.33 g, 8.12 mmol) butan-2-yl)formamide as a 50 : 50 mixture rotamers, as a white
1
was added as a solid in one portion to a solution of cesium solid (0.16 g, 97%). H NMR (400 MHz; CDCl ) δ 0.89 (t, J = 6.9
3
carbonate (2.91 g, 8.25 mmol) in dry DMF (12 mL) at room Hz, 3H, both rotamers), 1.28–1.46 (m, 20H), 1.36 (s, 6H, one
temperature. The solution was stirred for 15 min, then rotamer), 1.40 (s, 6H, one rotamer), 1.72–1.82 (m, 8H, both
1
-bromoheptane (1.3 mL, 8.27 mmol) was added dropwise to rotamers), 1.99–2.03 (m, 2H, one rotamer), 2.52–2.60 (m, 4H),
the now bright yellow solution. The solution was stirred at 3.91 (t, J = 6.6 Hz, 2H, one rotamer), 3.92 (t, J = 6.6 Hz, 2H, one
room temperature for 24 h. The suspension was poured onto rotamer), 5.25 (br s, 1H, one rotamer), 5.98 (br d, J = 12.0 Hz,
2
M aqueous hydrochloric acid solution and extracted with ethyl one rotamer), 6.79–6.83 (m, 4H), 7.05–7.09 (m, 4H), 8.05 (d, J =
acetate (×3). The organic extracts were combined and washed 2.0 Hz, 1H, one rotamer), 8.28 (d, J = 12.0 Hz, 1H, one
1
3
with water (×2) and brine, then dried (Na
2 4 3
SO ). The solvent rotamer); C NMR (100 MHz; CDCl ) δ 14.2, 22.7, 26.1, 27.3,
was removed under reduced pressure and the crude material 28.8, 29.18, 29.19, 29.43, 29.45, 29.51, 29.8, 31.9, 42.6, 46.1,
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1
2.9, 54.1, 68.16, 68.17, 114.6, 114.7, 129.2, 129.3, 133.2, 133.9, room temperature. The THF was removed under reduced
−
1
57.5, 157.7, 160.6, 163.2; IR (NaCl, neat) 1683, 3290 cm
;
pressure and the residue extracted with dichloromethane (×4).
Na [M + Na] 328.2252, The organic extracts were combined and dried (Na SO ). The
solvent was removed under reduced pressure to afford a yellow
+
HRMS (ESI-MS): m/z calcd for C19
found 328.2247.
H
31
O
2
2
4
1
(b) (2S)-N-(4-(4-heptyloxyphenyl)-1-hydroxy-2-methylbutan- oil. H NMR analysis showed the diastereometric ratio to be
2
-yl)formamide (56 mg, 0.18 mmol) in 6 M aqueous hydro- 70 : 30, in favour of the starting material, through integration
chloric acid solution (9 mL) was heated at reflux for 16 h. The of the benzylic multiplets at δ 2.46–2.62 and δ 2.65–2.81
solution was cooled to room temperature and basified to pH respectively. The mixture was purified by flash chromatography
1
4 using 2 M aqueous sodium hydroxide solution. The suspen- on silica gel, eluting with 3% ethyl acetate/n-hexane, to afford
sion was extracted with dichloromethane (×3). The organic the starting material as a clear colourless oil (89 mg, 59%). All
1
0
extracts were combined and dried (Na SO ). The solvent was spectroscopic data matched those reported previously.
2
4
removed under reduced pressure to afford the title compound Further elution afforded (2S,5S)-5-isopropyl-3,6-dimethoxy-2-
1
1
4 as a clear colourless oil (49 mg, 96%). H NMR (300 MHz; (4′-t-butyldimethyl-silyloxyphenethyl)-2,5-dihydropyrazine as a
2
5.0
1
CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), 1.16 (s, 6H), 1.29–1.46 (m, clear colourless oil (41 mg, 27%). [α]
= −6 (0.5, CHCl ); H
3
D
3
1
3
3
0H), 1.61–1.67 (m, 2H), 1.71–1.81 (m, 2H) 2.56–2.61 (m, 2H), NMR (300 MHz; CDCl ) δ 0.18 (s, 6H), 0.75 (d, J = 6.8 Hz, 3H),
.92 (t, J = 6.6 Hz, 2H), 6.79–6.84 (m, 2H), 7.07–7.12 (m, 2H); 0.98 (s, 9H), 1.08 (d, J = 6.8 Hz, 3H), 1.68–1.80 (m, 1H),
1
3
C NMR (75 MHz; CDCl ) δ 14.2, 22.7, 26.2, 29.2, 29.5, 30.3, 2.12–2.29 (m, 2H), 2.65–2.81 (m, 2H), 3.68 (s, 3H), 3.71 (s, 3H),
3
3
0.5, 31.9, 47.5, 49.6, 68.2, 114.6, 129.2, 134.7, 157.4; IR (NaCl, 3.94–3.03 (m, 2H), 6.73–6.78 (m, 2H), 7.05–7.10 (m, 2H);
−
1
13
neat) 3189, 3284, 3353 cm ; HRMS (ESI-MS): m/z calcd for
C H NO [M + H] 278.2484, found 278.2480.
C NMR (75 MHz; CDCl
31.6, 37.6, 52.38, 52.41, 55.2, 61.1, 120.0, 129.5, 135.0, 153.8,
(18). 163.2, 163.9; IR (NaCl, neat) 1697 cm ; HRMS (ESI-MS): m/z
3
) δ −4.3, 17.7, 18.3, 19.7, 25.9, 31.4,
+
1
8
32
−1
(
2S)-4-(4′-Heptyloxyphenyl)-2-methylbutane-1,2-diol
+
Potassium osmate dihydrate (1 mg, 2.71 μmol) was added as a calcd for C23
39 2 3
H N O Si [M + Na] 419.2729, found 419.2714.
solid in one portion to a suspension of 1-(heptyloxy)-4-(3′- (b) Cesium fluoride (0.20 g, 1.33 mmol) was added as a
1
1
methylbut-3′-enyl)benzene (0.13 g, 0.48 mmol), potassium solid in one portion to a solution of (2S,5S)-5-isopropyl-3,6-
carbonate (0.20 g, 1.45 mmol), potassium ferricyanide (0.48 g, dimethoxy-2-(4′-t-butyldimethylsilyloxyphenethyl)-2,5-dihydro-
1
.45 mmol) and (DHQD) PHAL (20 mg, 24 μmol) in 1 : 1 pyrazine (0.28 g, 0.67 mmol) in dry DMF (5 mL) at room temp-
2
t-butanol : water (8 mL) at 0 °C. The solution was allowed to erature. The solution was stirred for 15 min, where it had
slowly warm to room temperature, in the cold bath, over 20 h. turned dark orange, before 1-bromoheptane (0.20 mL,
Sodium sulfite (0.55 g, 4.35 mmol) was added and the solution 1.27 mmol) was added dropwise. The solution was stirred at
allowed to stir for a further 15 min. The mixture was diluted room temperature for 17 h. Water was added and the mixture
with water and extracted with ethyl acetate (×3). The organic was extracted with ethyl acetate (×3). The organic extracts were
extracts were combined and washed with brine, then dried combined and washed with water and brine, then dried
2 4 2 4
(Na SO ). The solvent was removed under reduced pressure (Na SO ). The solvent was removed under reduced pressure
and the crude material purified by flash chromatography on and the crude material purified by flash chromatography on
silica gel, eluting with 60% ethyl acetate/n-hexane, to afford silica gel, eluting with 2% ethyl acetate/n-hexane, to afford
the title compound 18 as a clear colourless oil (0.12 g, 83%). (2S,5S)-5-isopropyl-3,6-dimethoxy-2-(4′-heptyloxyphenethyl)-
2
1.0
1
[α]
D
= −4 (0.5, CHCl
3 3
); H NMR (300 MHz; CDCl ) δ 0.91 (t, 2,5-dihydropyrazine as a clear colourless oil (0.19 g, 72%).
2
5.0
1
J = 6.8 Hz, 3H), 1.22 (s, 3H), 1.32–1.48 (m, 8H), 1.73–1.82 (m, [α]
= +44 (0.5, CHCl ); H NMR (300 MHz; CDCl ) δ 0.74 (d,
D
3
3
4
H), 2.58–2.67 (m, 2H), 2.94 (br s, 1H), 3.29 (br s, 1H), 3.42 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 6.8 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H),
J = 11.0 Hz, 1H), 3.50 (d, J = 11.0 Hz, 1H), 3.91 (t, J = 6.6 Hz, 1.25–1.49 (m, 8H), 1.70–1.81 (m, 3H), 2.12–2.29 (m, 2H),
1
3
2
H), 6.79–6.83 (m, 2H), 7.08–7.11 (m, 2H); C NMR (75 MHz; 2.66–2.81 (m, 2H), 3.68 (s, 3H), 3.71 (s, 3H), 3.92 (t, J = 6.6 Hz,
CDCl ) δ 14.2, 22.7, 23.2, 26.1, 29.16, 29.21, 29.4, 31.9, 40.7, 2H), 3.94–4.02 (m, 2H), 6.79–6.84 (m, 2H), 7.11–7.15 (m, 2H);
3
1
3
6
3
8.1, 69.8, 73.1, 114.6, 129.2, 134.2, 157.4; IR (NaCl, neat)
352 cm ; HRMS (ESI-MS): m/z calcd for C H O Na [M + 29.5, 31.4, 31.5, 31.9, 37.7, 52.39, 52.42, 55.2, 61.0, 68.2, 114.5,
Na] 317.2093, found 317.2093.
Synthesis of (2S)-2-amino-4-(4′-heptyloxyphenyl)butan-1-ol HRMS (ESI-MS): m/z calcd for C24
19). (a) A solution of n-butyllithium in hexanes (0.17 mL, 2.1 found 403.2959.
C NMR (75 MHz; CDCl
3
) δ 14.2, 17.7, 19.7, 22.8, 26.2, 29.2,
−1
1
8
30 3
+
−1
129.5, 134.2, 157.5, 163.2, 163.9; IR (NaCl, neat) 1693 cm ;
+
39 2 3
H N O [M + H] 403.2961,
(
M, 0.36 mmol) was added dropwise to a solution of (2R,5S)-5-
(c) A solution of TFA (2.5 mL) in water (5 mL) was added
isopropyl-3,6-dimethoxy-2-(4′-t-butyldimethylsilyloxyphenethyl)- dropwise to a solution of (2S,5S)-5-isopropyl-3,6-dimethoxy-2-
1
0
2
,5-dihydropyrazine (0.15 g, 0.35 mmol) in freshly distilled (4′-heptyloxyphenethyl)-2,5-dihydropyrazine (0.19 g, 0.48
THF (2 mL) at −78 °C (dry ice/acetone). The solution was mmol) in acetonitrile (12 mL). The solution was stirred at
stirred at −78 °C for 15 min after which, it had turned dark room temperature for 4 h after which the acetonitrile was
yellow. Methanol (28 μL, 0.69 mmol) was added dropwise and removed under reduced pressure. The residue was diluted with
the solution allowed to warm slowly, in the cold bath, to water and neutralised with portions of solid sodium bicarbon-
−40 °C over 3 h. The reaction was quenched with saturated ate, then extracted with dichloromethane (×4). The organic
aqueous sodium bicarbonate solution and allowed to warm to extracts were combined and dried (Na SO ). The solvent was
2
4
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2
4.3
1
removed under reduced pressure and the crude material puri- ourless oil (0.37 g, 87%). [α]
D
3
= + 36 (0.5, CHCl ); H NMR
fied by flash chromatography on silica gel, eluting with ethyl (300 MHz; CDCl ) δ 0.17 (s, 6H), 0.66 (t, J = 7.4 Hz, 3H), 0.71
3
acetate, to afford methyl (2S)-2-amino-4-(4′-heptyloxyphenyl) (d, J = 6.8 Hz, 3H), 0.97 (s, 9H), 1.12 (d, J = 6.8 Hz, 3H),
2
2.3
butanoate as a clear colourless oil (0.15 g, 99%). [α]
D
= +14 1.51–1.63 (m, 1H), 1.75–1.90 (m, 2H), 2.09 (td, J = 12.8, 4.2 Hz,
1
(
0.5, CHCl ); H NMR (300 MHz; CDCl ) δ 0.88 (t, J = 6.8 Hz, 1H), 2.21 (td, J = 12.8, 4.2 Hz, 1H), 2.32–2.46 (m, 2H), 3.71 (s,
3
3
3
1
1
7
2
1
H), 1.26–1.46 (m, 8H), 1.53 (br s, 2H), 1.70–1.86 (m, 3H), 3H), 3.72 (s, 3H), 3.93 (d, J = 3.3 Hz, 1H), 6.71–6.75 (m, 2H),
1
3
.97–2.08 (m, 1H), 2.57–2.73 (m, 2H), 3.43 (dd, J = 7.9, 5.2 Hz, 6.98–7.02 (m, 2H); C NMR (75 MHz; CDCl
3
) δ −4.3, 8.4, 17.2,
H) 3.69 (s, 3H), 3.91 (t, J = 6.6 Hz, 2H), 6.78–6.83 (m, 2H), 18.4, 19.7, 25.9, 30.78, 30.81, 34.0, 42.4, 52.3, 52.4, 60.9, 62.7,
1
3
.06–7.11 (m, 2H); C NMR (75 MHz; CDCl
3
) δ 14.1, 22.7, 26.1, 119.9, 129.3, 135.5, 153.6, 162.9, 164.1; IR (NaCl, neat)
−
1
9.1, 29.4, 31.1, 31.9, 36.7, 52.0, 53.9, 68.1, 114.5, 129.4, 133.1, 1691 cm ; HRMS (ESI-MS): m/z calcd for C25
57.6, 176.6; IR (NaCl, neat) 1738, 3317, 3382 cm ; HRMS + Na] 469.2862, found 469.2845.
42 2 3
H N O SiNa [M
−
1
+
+
(ESI-MS): m/z calcd for C18
H
29NO
3
Na [M + Na] 330.2045,
(b) Cesium fluoride (0.12 g, 0.77 mmol) was added as a
found 330.2031.
solid in one portion to a solution of (2S,5S)-5-isopropyl-3,6-
(d) Lithium aluminium hydride (37 mg, 0.98 mmol) was dimethoxy-2-(4′-t-butyldimethylsilyloxyphenethyl)-2-ethyl-2,5-
added as a solid, in one portion, to a solution of methyl (2S)-2- dihydropyrazine (0.17 g, 0.39 mmol) in dry DMF (3.5 mL) at
amino-4-(4′-heptyloxyphenyl)butanoate (0.15 g, 0.48 mmol) in room temperature. The solution was stirred for 15 min, where
freshly distilled THF (4 mL) at 0 °C. The solution was stirred at it had turned dark orange, before 1-bromoheptane (90 μL,
0
°C for 20 min then the cold bath was removed and the solu- 0.44 mmol) was added dropwise. The solution was stirred at
tion stirred at room temperature for 2 h. The reaction was room temperature for 17 h. Water was added and the mixture
quenched with saturated aqueous sodium sulfate solution and was extracted with ethyl acetate (×3). The organic extracts were
the mixture was extracted with ethyl acetate (×4). The organic combined and washed with water and brine, then dried
extracts were combined and washed with saturated aqueous (Na
sodium bicarbonate solution, water and brine, then dried and the crude material purified by flash chromatography on
Na SO ). The solvent was removed under reduced pressure silica gel, eluting with 3% ethyl acetate/n-hexane, to afford
2 4
SO ). The solvent was removed under reduced pressure
(
2
4
and the crude material purified by flash chromatography on (2S,5S)-5-isopropyl-3,6-dimethoxy-2-(4′-heptyloxyphenethyl)-2-
silica gel, eluting with 3% methanol/3% triethylamine/dichloro- ethyl-2,5-dihydropyrazine as a clear colourless oil (0.14 g,
2
4.8
1
methane, to afford the title compound 19 as a clear gum 84%). [α]
D
= + 30 (0.5, CHCl
); H NMR (300 MHz; 0.66 (t, J = 7.4 Hz, 3H), 0.71 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 6.8
CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), 1.26–1.46 (m, 8H), 1.51–1.63 Hz, 3H), 1.12 (d, J = 6.8 Hz, 3H), 1.31–1.47 (m, 8H), 1.51–1.63
3 3
); H NMR (300 MHz; CDCl ) δ
2
6.4
1
(59 mg, 44%). [α]
D
= −2 (0.5, CHCl
3
3
(m, 1H), 1.69–1.81 (m, 3H), 2.07 (br s, 1H), 2.17 (br s, 2H), (m, 1H), 1.71–1.90 (m, 4H), 2.09 (td, J = 12.8, 4.2 Hz, 1H), 2.22
2
1
6
.54–2.73 (m, 2H), 2.83–2.91 (m, 1H), 3.32 (dd, J = 10.6, 7.8 Hz, (td, J = 12.8, 4.2 Hz, 1H), 2.33–2.47 (m, 2H), 3.71 (s, 3H), 3.72
H), 3.61 (dd, J = 10.6, 3.5 Hz, 1H), 3.91 (t, J = 6.6 Hz, 2H), (s, 3H), 3.90–3.94 (m, 3H), 6.79–6.82 (m, 2H), 7.04–7.07 (m,
1
3
13
.69–6.83 (m, 2H), 7.06–7.09 (m, 2H); C NMR (75 MHz; 2H); C NMR (75 MHz; CDCl
3
) δ 8.4, 14.2, 17.2, 19.7, 22.8,
CDCl
3
) δ 14.2, 22.8, 26.2, 29.2, 29.5, 31.6, 31.9, 36.3, 52.5, 66.6, 26.2, 29.2, 29.5, 30.7, 30.8, 32.0, 34.0, 42.6, 52.3, 52.4, 60.9,
6
3
2
8.2, 114.7, 129.3, 133.6, 157.6; IR (NaCl, neat) 3120, 3280, 62.7, 68.2, 114.5, 129.3, 134.8, 157.3, 162.9, 164.2; IR (NaCl,
339 cm− ; HRMS (ESI-MS): m/z calcd for C17
1
H
30NO
[M + H]
+
neat) 1691 cm ; HRMS (ESI-MS): m/z calcd for C26
[M + H] 431.3274, found 431.3261.
−1
H
N
O
2
43
2
3
+
80.2277, found 280.2265.
Synthesis of (2S)-2-amino-2-ethyl-4-(4′-heptyloxyphenyl)
butan-1-ol (20). (a) A solution of n-butyllithium in hexanes dropwise to a solution of (2S,5S)-5-isopropyl-3,6-dimethoxy-2-
0.47 mL, 2.1 M, 0.99 mmol) was added dropwise to a solution (4′-heptyloxyphenethyl)-2-ethyl-2,5-dihydropyrazine (0.14 g,
of (2R,5S)-5-isopropyl-3,6-dimethoxy-2-(4′-t-butyldimethyl- 0.33 mmol) in acetonitrile (6 mL). The solution was stirred at
(c) A solution of TFA (3 mL) in water (6 mL) was added
(
1
0
silyloxyphenethyl)-2,5-dihydropyrazine (0.39 g, 0.94 mmol) room temperature for 76 h after which the acetonitrile was
in freshly distilled THF (6 mL) at −78 °C (dry ice/acetone). The removed under reduced pressure. The residue was diluted with
solution was stirred at −78 °C for 15 min after which, it had water and neutralised with portions of solid sodium bicarbon-
turned dark yellow. Ethyl iodide (83 μL, 1.03 mmol) was added ate, then extracted with dichloromethane (×4). The organic
2 4
dropwise and the solution allowed to warm slowly, in the cold extracts were combined and dried (Na SO ). The solvent was
bath, to −10 °C over 5 h. The reaction was quenched with satu- removed under reduced pressure and the crude material puri-
rated aqueous sodium bicarbonate solution and allowed to fied by flash chromatography on silica gel, eluting with 50%
warm to room temperature. The solvent was removed under ethyl acetate/n-hexane, to afford methyl (2S)-2-amino-2-ethyl-4-
reduced pressure and the residue extracted with dichloro- (4′-heptyloxyphenyl)butanoate as a clear colourless oil (27 mg,
2
7.8
1
methane (×4). The organic extracts were combined and dried 25%). [α]
D
3 3
= +20 (0.5, CHCl ); H NMR (400 MHz; CDCl )
(
Na SO ). The solvent was removed under reduced pressure 0.84–0.90 (m, 6H), 1.25–1.37 (m, 6H), 1.40–1.47 (m, 2H),
2
4
and the crude material purified by flash chromatography 1.56–1.65 (m, 1H), 1.69 (br s, 2H), 1.71–1.86 (m, 4H), 1.99–2.06
on silica gel, eluting with 3% ethyl acetate/n-hexane, to (m, 1H), 2.39 (td, J = 12.6, 4.8 Hz, 1H), 2.62 (td, J = 12.6, 5.3
afford (2S,5S)-5-isopropyl-3,6-dimethoxy-2-(4′-t-butyldimethyl- Hz, 1H), 3.71 (s, 3H), 3.91 (t, J = 6.6 Hz, 2H), 6.79–6.82 (m, 2H),
1
3
silyloxyphenethyl)-2-ethyl-2,5-dihydropyrazine as a clear col- 7.05–7.08 (m, 2H); C NMR (100 MHz; CDCl ) δ 8.4, 14.2, 22.7,
3
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2
1
3
3
6.1, 29.2, 29.4, 29.8, 31.9, 33.2, 42.0, 52.2, 61.7, 68.1, 114.6, 23.3 μmol) in n-propanol (2.1 mL) and a solution of 1-(heptyl-
1
1
29.3, 133.6, 157.5, 177.6; IR (NaCl, neat) 1732, 3325, oxy)-4-(3′-methylbut-3′-enyl)benzene (0.12 g, 0.47 mmol) in
385 cm− ; HRMS (ESI-MS): m/z calcd for C20
1
H
+
n-propanol (0.4 mL). The solution was stirred vigorously at
3
34NO [M + H]
36.2539, found 336.22530.
room temperature until it became homogeneous, then potass-
(
d) Lithium aluminium hydride (5 mg, 0.13 mmol) was ium osmate dihydrate (7 mg, 19.0 μmol) was added as a solid
added as a solid in one portion to a solution of methyl (2S)-2- in one portion. The solution immediately turned dark green
amino-2-ethyl-4-(4′-heptyloxyphenyl)butanoate (27 mg, and was stirred at room temperature for 24 h after which, it
0.5 μmol) in freshly distilled THF (0.5 mL) at 0 °C. The solu- had turned light yellow. Sodium sulphite (0.59 g, 4.70 mmol)
8
tion was stirred at 0 °C for 20 min then the cold bath was was added and the suspension was stirred for a further
removed and the solution stirred at room temperature for 1 h. 10 min. The mixture was diluted with water and extracted with
The reaction was quenched with saturated aqueous sodium ethyl acetate (×3). The organic extracts were combined and
sulfate solution and the mixture was extracted with ethyl washed with brine, then dried (Na SO ). The solvent was
2 4
acetate (×4). The organic extracts were combined and washed removed under reduced pressure and the crude material was
with saturated aqueous sodium bicarbonate solution, water purified by flash chromatography on silica gel, eluting with
and brine, then dried (Na SO ). The solvent was removed dichloromethane, to remove any residual 2-(trimethylsilyl)
2 4
under reduced pressure and the crude material purified by ethyl carbamate, then 30% ethyl acetate/n-hexane, to afford
flash chromatography on silica gel, eluting with 1% methanol/ 2-(trimethylsilyl)ethyl (2S)-(4-(4-heptyloxyphenyl)-1-hydroxy-2-
3
% triethylamine/dichloromethane, to afford the title com- methylbutan-2-yl)carbamate as a clear colourless oil (0.16 g,
2
6.9
21.0
1
pound 20 as a clear gum (15 mg, 60%). [α]
CHCl3); H NMR (300 MHz; CDCl ) 0.86–0.93 (m, 6H), 0.04 (s, 9H), 0.89 (t, J = 6.8 Hz, 3H), 0.95–1.01 (m, 2H), 1.23 (s,
D
= −2 (0.5, 77%). [α]
D
3 3
= −2 (0.5, CHCl ); H NMR (300 MHz; CDCl ) δ
1
3
1
2
6
.25–1.60 (m, 12H), 1.64–1.81 (m, 3H), 1.89 (br s, 2H), 3H), 1.26–1.47 (m, 10H), 1.72–1.81 (m, 2H), 2.23 (br s, 1H),
.47–2.58 (m, 2H), 2.37 (s, 2H), 3.92 (t, J = 6.6 Hz, 2H), 2.61–2.67 (m, 2H), 3.15–3.28 (m, 2H), 3.92 (t, J = 6.6 Hz, 2H),
1
3
.79–6.83 (m, 2H), 7.06–7.11 (m, 2H); C NMR (75 MHz; 4.14–4.19 (m, 2H), 5.01 (br s, 1H), 6.80–6.83 (m, 2H), 7.08–7.10
1
3
3 3
CDCl ) δ 7.9, 14.2, 22.7, 26.2, 29.1, 29.17, 29.20, 29.5, 31.9, (m, 2H); C NMR (75 MHz; CDCl ) δ −1.34, 14.2, 17.9, 22.7,
3
3
3
8.7, 55.3, 68.0, 68.2, 114.6, 129.2, 134.3, 157.5; IR (NaCl, neat) 24.7, 26.2, 29.2, 29.3, 29.5, 31.9, 42.1, 50.6, 63.5, 68.2, 77.4,
355 cm− ; HRMS (ESI-MS): m/z calcd for C H NO [M + H]
1
+
114.7, 129.3, 134.1, 157.5, 157.9; IR (NaCl, neat) 1699,
1
9
34
2
−1
08.2589, found 308.2572.
4S)-4-(4′-Heptyloxyphenethyl)-4-methyloxazolidin-2-one (22). [M + Na] 460.2859, found 460.2860.
2S)-t-Butyl(1-hydroxy-4-(4′-heptyloxyphenyl)-2-methylbutan-2-yl) (b) A solution of hydrochloric acid in diethyl ether
carbamate (22 mg, 49 μmol), potassium hydroxide (11 mg, (0.53 mL, 2 M, 1.06 mmol) was added dropwise to a solution
.20 mmol) and tosyl chloride (11 mg, 58 μmol) in diethyl 2-(trimethylsilyl)ethyl (2S)-(4-(4-heptyloxyphenyl)-1-hydroxy-2-
4
3417 cm ; HRMS (ESI-MS): m/z calcd for C24H43NO SiNa
+
(
(
1
0
0
ether (2 mL) was heated at reflux for 15 h. The solution was methylbutan-2-yl)carbamate (92 mg, 0.21 mmol) in methanol
allowed to cool to room temperature and poured onto ice (5 mL) at room temperature. The solution was stirred at room
water. The mixture was extracted with diethyl ether (×3). The temperature for 5 h. then the solvent was removed under
organic extracts were combined and washed with water and reduced pressure. The residue was dissolved in 1 M aqueous
2 4
brine, then dried (Na SO ). The solvent was removed under hydrochloric acid and extracted with diethyl ether (×3). The
reduced pressure and the crude material purified by flash remaining aqueous layer was collected and neutralised with
chromatography on silica gel, eluting with 20% ethyl acetate/ solid sodium bicarbonate and extracted with dichloromethane
n-hexane, to afford the title compound 22 as a clear colourless (×3). The organic extracts were combined and washed with
2
6.7
1
gum (14 mg, 90%). [α]
D
3 2 4
= +12 (0.5, CHCl ); H NMR brine, then dried (Na SO ). The solvent was removed under
(
300 MHz; CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), 1.26–1.47 (m, 8H), reduced pressure and the crude material purified by flash
3
1
.39 (s, 3H), 1.71–1.81 (m, 2H), 1.83–1.89 (m, 2H), 2.58–2.63 chromatography on silica gel, eluting with 1% triethylamine/
(
(
(
2
1
m, 2H), 3.91 (t, J = 6.6 Hz, 2H), 4.04 (d, J = 8.5 Hz, 1H), 4.15 5% methanol/dichloromethane, to afford the title compound
2
5.6
d, J = 8.5 Hz, 1H), 6.48 (br s, 1H), 6.78–6.83 (m, 2H), 7.04–7.08 23 as a clear colourless gum (28 mg, 45%). [α]
m, 2H); C NMR (75 MHz; CDCl ) δ 14.2, 22.7, 25.9, 26.1, CHCl ); H NMR (300 MHz; CDCl
3 3 3
= −2 (0.5,
) δ 0.89 (t, J = 6.5 Hz, 3H),
9.2, 29.4, 29.5, 31.9, 42.6, 57.8, 68.1, 75.8, 114.7, 129.2, 132.7, 1.20 (s, 3H), 1.26–1.44 (m, 8H), 1.71–1.80 (m, 4H), 2.61–2.71
57.7, 159.6; IR (NaCl, neat) 1729, 1764, 3161, 3248 cm−1
(m, 4H), 2.95 (br s, 3H), 3.91 (t, J = 6.5 Hz, 2H), 6.79–6.82 (m,
D
1
3
1
;
+
13
HRMS (ESI-MS): m/z calcd for C19
H
29NO
3
Na [M + Na]
3
2H), 7.08–7.11 (m, 2H); C NMR (75 MHz; CDCl ) δ 14.2, 22.7,
3
42.2045, found 342.2045.
Synthesis of (2S)-1-amino-4-(4′-heptyloxyphenyl)-2-methyl- 129.3, 134.4, 157.4; IR (NaCl, neat) 3298, 3362 cm ; HRMS
24.3, 26.2, 29.2, 29.3, 29.5, 31.9, 42.0, 50.7, 68.2, 71.4, 114.6,
−
1
+
butan-2-ol (23). (a) A solution of aqueous sodium hydroxide (ESI-MS): m/z calcd for C18
H
32NO
2
[M + H] 294.2433, found
(3.5 mL, 0.4 M, 1.40 mmol) was added dropwise to a solution 294.2432.
of 2-(trimethylsilyl)ethyl carbamate (0.23 g, 1.40 mmol) in
n-propanol (1 mL) at room temperature. 1,3-Dichloro-5,5-di- diol (O-FTY720 (24)). (a) Cesium fluoride (0.32 g, 2.09 mmol)
Synthesis of 2-amino-2-(4′-heptyloxyphenethyl)propane-1,3-
8
b
methylhydantoin (0.18 g, 0.93 mmol) was added as a solid in was added as a solid in one portion to a solution of diethyl
1
7
one portion, followed by a solution of (DHQD) AQN (20 mg, 2-acetamido-2-(4′-(t-butyldimethylsilyloxy)phenethyl)malonate
2
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(
26) (0.63 g, 1.39 mmol) in dry DMF (14 mL) at room tempera- The solution was stirred at 0 °C for 1 hour then quenched with
ture. The solution was stirred for 15 min, whereupon it turned saturated aqueous sodium bicarbonate solution. The mixture
dark orange, before 1-bromoheptane (0.24 mL, 1.53 mmol) was extracted with dichloromethane (×3). The organic extracts
was added dropwise. The solution was stirred at room temp- were combined and washed with saturated aqueous sodium
erature for 17 h. Water was added and the mixture was bicarbonate solution, water and brine, then dried (Na SO ).
2
4
extracted with ethyl acetate (×3). The organic extracts were The solvent was removed under reduced pressure and the
combined and washed with water and brine, then dried crude material was purified by flash chromatography on silica
(
Na SO ). The solvent was removed under reduced pressure gel, eluting with 3% ethyl acetate/n-hexane, to afford the title
2 4
2
5.3
and the crude material purified by flash chromatography on compound 28 as a clear colourless oil (56 mg, 46%). [α]
D
= +
) δ 0.69 (d, J = 6.8
2-acetamido-2-(4′-(t-butyldimethylsilyloxy)phenethyl) Hz, 3H), 1.12 (d, J = 6.8 Hz, 3H), 1.31 (s, 3H), 1.87 (td, J = 12.8,
malonate as a white solid (0.57 g, 94%) with all the analytical 5.0 Hz, 1H), 2.10 (td, J = 12.8, 4.6 Hz, 1H), 2.28–2.43 (m, 2H),
1
silica gel, eluting with 50% ethyl acetate/n-hexane, to afford 48 (0.5, CHCl
diethyl
3 3
); H NMR (300 MHz; CDCl
1
7
data matching that reported in the literature. Mp 79–81 °C; 2.51 (td, J = 12.8, 5.0 Hz, 1H), 3.69 (s, 3H), 3.71 (s, 3H), 3.94 (d,
1
13
H NMR (300 MHz; CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H), 1.24 (t, J = J = 3.3 Hz, 1H), 7.14–7.16 (m, 2H), 7.21–7.26 (m, 2H); C NMR
3
7
2
4
.3 Hz, 6H), 1.28–1.48 (m, 8H), 1.71–1.80 (m, 2H), 1.99 (s, 3H), (75 MHz; CDCl
.39–2.44 (m, 2H), 2.62–2.68 (m, 2H), 3.90 (t, J = 6.6 Hz, 2H), 52.48, 58.3, 60.5, 118.9 (q, JC–F = 318.9 Hz), 121.2, 130.2, 143.5,
3
) δ 17.0, 19.7, 28.6, 30.6, 31.1, 42.3, 52.46,
−
1
.16–4.24 (m, 4H), 6.76 (br s, 1H), 6.78–6.82 (m, 2H), 7.01–7.06 147.8, 162.5, 165.4; IR (NaCl, neat) 1692 cm ; HRMS (ESI-MS):
1
3
+
(
2
1
m, 2H); C NMR (75 MHz; CDCl
3
) δ 14.1, 14.2, 22.8, 23.2, m/z calcd for C19
6.1, 29.2, 29.37, 29.44, 31.9, 33.7, 62.7, 66.5, 68.2, 114.6, 451.1451.
29.4, 132.5, 157.7, 168.2, 169.1. Synthesis of (2S)-2-amino-2-methyl-4-(4′-octylphenyl)butan-
b) Lithium aluminium hydride (78 mg, 2.06 mmol) 1-ol (25). (a) A solution of n-butyllithium in hexanes (1.2 mL,
was added as a solid in one portion to a solution of diethyl 1.4 M, 1.64 mmol) was added dropwise to a solution of freshly
-acetamido-2-(4′-(t-butyldimethylsilyloxy)phenethyl)malonate distilled (S)-Schöllkopf’s reagent 29 (0.31 g, 1.64 mmol) in
0.36 g, 0.84 mmol) in freshly distilled THF (4 mL) at 0 °C. The freshly distilled THF (3.2 mL) at −78 °C (dry ice/acetone). The
solution was stirred at 0 °C for 20 min then the cold bath was solution was stirred at −78 °C for 15 min, where it had turned
26 3 2 5
H F N O S [M + H] 451.1515, found
(
2
(
1
1
removed and the solution stirred at room temperature for 1 h. dark yellow. A solution of 1-(2-iodoethyl)-4-octylbenzene (30)
The reaction was quenched with saturated aqueous sodium (0.53 g, 1.55 mmol) in freshly distilled THF (3.2 mL) at −78 °C
sulfate solution and the mixture was extracted with ethyl was added dropwise. The solution was stirred for a further
acetate (×4). The organic extracts were combined and washed 30 min at −78 °C then allowed to slowly warm to −15 °C over
with saturated aqueous sodium bicarbonate solution, water 4 h. The reaction mixture was quenched with saturated
and brine, then dried (Na SO ). The solvent was removed aqueous sodium bicarbonate solution and allowed to warm to
2 4
under reduced pressure and the crude material used in the room temperature. The THF was removed under reduced
next step without any further purification. The crude material pressure and the residue extracted with dichloromethane (×4).
2 4
was dissolved in ethanol (4 mL) and concentrated hydrochloric The organic extracts were combined and dried (Na SO ). The
acid solution (32%, 1 mL) was added dropwise. The mixture solvent was removed under reduced pressure and the crude
was heated at reflux for 16 h. The solution was allowed to cool material was purified by flash chromatography on silica gel,
to room temperature and the ethanol was removed under eluting with 2% ethyl acetate/n-hexane, to afford (2R,5S)-5-iso-
reduced pressure. The residue was neutralised with solid propyl-3,6-dimethoxy-2-(4-octylphenethyl)-2,5-dihydro-pyrazine
2
6.8
sodium bicarbonate and extracted with ethyl acetate (×4). The as a clear colourless oil (0.52 g, 83%). [α]
D
= −8 (0.5, CHCl
) δ 0.70 (d, J = 6.8 Hz, 3H), 0.88 (t, J =
dried (Na SO ). The solvent was removed under reduced 6.6 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 1.27–1.35 (m, 10H),
3
);
1
organic extracts were combined and washed with brine, then
H NMR (300 MHz; CDCl
3
2
4
pressure to afford a brown gum which was recrystallised from 1.54–1.61 (m, 2H), 1.93–2.05 (m, 1H), 2.10–2.21 (m, 1H),
ethanol to afford the title compound 24 as a white solid 2.23–2.33 (m, 1H), 2.50–2.66 (m, 4H), 3.70 (s, 6H), 3.97 (t, J =
(
0.16 g, 62%) with all the analytical data matching that 3.5 Hz, 1H), 4.06 (dt, J = 6.8, 3.5 Hz, 1H), 7.06–7.12 (m, 4H);
8
1
13
reported in the literature. H NMR (300 MHz; MeOD) δ 0.91
t, J = 6.7 Hz, 3H), 1.33–1.50 (m, 8H), 1.65–1.76 (m, 4H), 29.6, 30.7, 31.8, 31.9, 32.0, 35.7, 36.0, 52.50, 52.53, 55.2, 61.0,
.55–2.61 (m, 2H), 3.48 (d, J = 11.0 Hz, 2H), 3.54 (d, J = 11.0 128.4, 128.5, 139.4, 140.4, 163.7, 163.9; IR (NaCl, neat)
3
C NMR (75 MHz; CDCl ) δ 14.3, 16.8, 19.2, 22.8, 29.4, 29.5,
(
2
Hz, 2H), 3.91 (t, J = 6.4 Hz, 2H), 6.78–6.81 (m, 2H), 7.10–7.13 1695 cm− ; HRMS (ESI-MS): m/z calcd for C25
1
+
41 2 2
H N O [M + H]
1
3
(m, 2H); C NMR (75 MHz; MeOD) δ 19.0, 26.7, 29.49, 29.53, 401.3168, found 401.3152.
2
1
9.6, 30.3, 66.4, 68.9, 69.5, 85.0, 115.47, 115.51, 130.2, 135.9,
58.9.
(b) A solution of n-butyllithium in hexanes (0.9 mL, 1.4 M,
1.30 mmol) was added dropwise to a solution of (2R,5S)-5-iso-
4
-(2-((2S,5S)-5-Isopropyl-3,6-dimethoxy-2-methyl-2,5-dihydro- propyl-3,6-dimethoxy-2-(4-octylphenethyl)-2,5-dihydropyrazine
pyrazin-2-yl)ethyl)phenyl
trifluoromethanesulfonate
(28). (0.52 g, 1.30 mmol) in freshly distilled THF (6.5 mL) at −78 °C
Triflic anhydride (0.05 mL, 0.30 mmol) was added dropwise to (dry ice/acetone). The solution was stirred at −78 °C for
1
0
a solution of phenol 27 (90 mg, 0.27 mmol) and pyridine 15 min, where it had turned dark yellow. Methyl iodide
(0.11 mL, 1.37 mmol) in dichloromethane (1.2 mL) at 0 °C. (0.16 mL, 2.57 mmol) was added dropwise. The solution was
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stirred for a further 30 min at −78 °C then allowed to slowly NMR (400 MHz; CDCl
3
) 0.88 (t, J = 7.0 Hz, 3H), 1.14 (s, 3H),
warm to −15 °C over 4 h. The reaction mixture was quenched 1.27–1.34 (m, 10H), 1.55–1.62 (m, 2H), 1.64–1.78 (m, 2H),
1
3
with saturated aqueous sodium bicarbonate solution and 2.54–2.63 (m, 7H), 3.34–3.42 (m, 2H), 7.07–7.12 (m, 4H);
allowed to warm to room temperature. The THF was removed NMR (125 MHz; CDCl ) δ 14.2, 22.7, 24.2, 29.3, 29.4, 29.5, 29.9,
under reduced pressure and the residue extracted with dichloro- 31.6, 31.9, 35.6, 41.7, 53.4, 69.7, 128.2, 128.5, 139.4, 140.4; IR
C
3
−
1
methane (×4). The organic extracts were combined and dried (NaCl, neat) 3266, 3333 cm ; HRMS (ESI-MS): m/z calcd for
+
(Na
2
SO
4
). The solvent was removed under reduced pressure
19
C H34NO [M + H] 292.2640, found 292.2625.
and the crude material was purified by flash chromatography
on silica gel, eluting with 2% ethyl acetate/n-hexane, to afford Biological data experimental
bis-lactim ether 31 as a clear colourless oil (0.28 g, 52%).
Cell lines. FDC.P1 mouse growth factor-dependent myeloid
cell line expressing empty vector (EV) (maintained in 25 units
per mL GM-CSF(GM)), and the oncogenic D816V mutant form
2
6.7
1
[
α]
= +45 (0.5, CHCl ); H NMR (500 MHz; CDCl ) δ 0.70 (d,
3 3
D
J = 6.8 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H), 1.12 (d, J = 6.8 Hz, 3H),
1
1
1
1
3
1
3
1
.27–1.33 (m, 10H), 1.31 (s, 3H), 1.55–1.62 (m, 2H), 1.88 (td, J =
3.0, 4.9 Hz, 1H), 2.11 (td, J = 13.0, 4.3 Hz, 1H), 2.28 (td, J =
3.0, 4.8 Hz, 1H), 2.35–2.41 (m, 1H), 2.46 (td, J = 13.0, 4.8 Hz,
H), 2.54–2.59 (m, 2H), 3.70 (s, 3H), 3.71 (s, 3H), 3.94 (d, J =
.3 Hz, 1H), 7.06–7.10 (m, 4H); C NMR (125 MHz; CDCl ) δ
4.3, 17.1, 19.7, 22.8, 28.7, 29.4, 29.5, 29.6, 30.7, 31.3, 31.8,
2.1, 35.7, 42.8, 52.4, 58.4, 60.5, 128.3, 128.4, 139.95, 140.33,
18
of human c-KIT (previously described by Frost ) were used to
5c
determine analog selectivity and capacity to activate PP2A.
The expression of c-KIT was routinely monitored by flow cyto-
18
metry. FDC.P1 cells were maintained in DMEM containing
1
3
3
10% fetal calf serum (FCS), 2 mM L-glutamine and 25 mM
HEPES.
Cell viability assays. Cell viability was determined following
treatment for 48 h with 0–10 μM of our novel analogs using a
−1
62.1, 165.7; IR (NaCl, neat) 1692 cm ; HRMS (ESI-MS): m/z
+
calcd for C26
c) A solution of TFA (3 mL) in water (6 mL) was added
dropwise to a solution of bis-lactim ether 31 (0.28 g,
.68 mmol) in acetonitrile (17 mL). The solution was stirred at
H
43
N
2
O
2
[M + H] 415.3325, found 415.3273.
5c
resazurin cell viability assay, as previously described.
(
PP2A activity. Cells were treated with 2.5 μM AAL(S) analogs
for 12 h. PP2A–C was immunoprecipitated from 150 μg protein
lysate with the Precipitator™ magnetic bead based platform
0
room temperature for 4 h after which the acetonitrile was
removed under reduced pressure. The residue was diluted with
water and neutralised with portions of solid sodium bicarbon-
ate, then extracted with dichloromethane (×4). The organic
extracts were combined and dried (Na SO ). The solvent was
(
(
Abnova) and phosphatase activity against a phosphopeptide
KRpTIRR) was determined as previously described.
5c
2
4
Acknowledgements
removed under reduced pressure and the crude material was
purified by flash chromatography on silica gel, eluting with This work was supported by a Cancer Council NSW grant
ethyl acetate, to afford methyl (2S)-2-amino-2-methyl-4-(4′- APP1030430. The authors would like to acknowledge funding
octylphenyl)butanoate as a clear colourless oil (0.14 g, 65%). from UNSW, Australia an Australian Postgraduate Award to
2
7.8
1
[α]
D
3 3
= +20 (0.5, CHCl ); H NMR (400 MHz; CDCl ) 0.88 (t, J = HDT, CTx Top-up scholarship to HDT. MDD and NMV are sup-
7
2
3
1
5
1
.0 Hz, 3H), 1.26–1.33 (m, 10H), 1.37 (s, 3H), 1.54–1.62 (m, ported by Cancer Institute fellowships. The authors thank Mr
H), 1.84–1.91 (m, 1H), 1.99–2.06 (m, 1H), 2.44–2.66 (m, 4H), Richard Kahl and. Nikita Panicker for technical support.
1
3
3
.70 (s, 3H), 7.06–7.10 (m, 4H); C NMR (100 MHz; CDCl ) δ
4.2, 22.8, 26.7, 29.4, 29.5, 29.6, 30. 4, 31.7, 32.0, 35.7, 43.0,
2.3, 57.9, 128.3, 128.6, 138.8, 140.7, 178.2; IR (NaCl, neat)
Notes and references
−
1
734, 3322, 3380 cm ; HRMS (ESI-MS): m/z calcd for
+
C H NO Na [M + Na] 342.2409, found 342.2389.
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2
0
33
2
(d) Lithium aluminium hydride (35 mg, 0.92 mmol) was
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2
5.2
1
solid (73 mg, 69%). Mp 93–95 °C; [α]
= +6 (0.5, CHCl ); H
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