An asymmetric synthesis of febrifugine, halofuginone and their hemiketal isomers

Article abstract of DOI:10.1016/j.tet.2017.07.041

A new synthesis of both enantiomers of naturally occurring febrifugine (1) and its analogue halofuginone (3) are reported. This robust route relies on an enzymatic kinetic resolution (EKR)-cross metathesis (CM) sequence, performed on allylic alcohol 8. A diastereoselective Lewis acid-mediated cyclisation of resultant enone 6 affords piperidine 12. In relation to its enantiomeric excess, values of 87 and 88% e.e were obtained for both enantiomers of 12 and for (?)-12 this was increased to 98% e.e by conducting the resolution process twice. A bromination (featuring an in situ temporary alcohol protection), alkylation and amino-deprotection sequence finally afforded the target compounds (+)- and (?)-1, and (+)- and (?)-3. Studies demonstrate that, as their ammonium salts, the compounds are stereochemically stable. However, as their free-bases, particularly in chloroform, C-2 isomerisation occurs. This isomerisation has been taken advantage of synthetically to provide enantioenriched (+)-isofebrifugine (2) and both enantiomers of isohalofuginone (14).

Full text of DOI:10.1016/j.tet.2017.07.041

Accepted Manuscript
An asymmetric synthesis of febrifugine, halofuginone and their hemiketal isomers
Shaun Smullen, Paul Evans
PII:
S0040-4020(17)30783-4
10.1016/j.tet.2017.07.041
TET 28873
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 10 May 2017
Revised Date: 11 July 2017
Accepted Date: 24 July 2017
Please cite this article as: Smullen S, Evans P, An asymmetric synthesis of febrifugine, halofuginone
and their hemiketal isomers, Tetrahedron (2017), doi: 10.1016/j.tet.2017.07.041.
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ACCEPTED MANUSCRIPT
An Asymmetric Synthesis of Febrifugine, Halofuginone and their Hemiketal Isomers
Shaun Smullen and Paul Evans
HO
R
N
3
2
HO
Br
Cl
N
O
3
2
HO
O
O
N
N
R'
N
H
N
H
HN
Cbz
(±)-8
O
R = R' = H, (+)-Febrifugine (1)
R = Br, R' = Cl, (+)-Halofuginone (3)
(−)-Halofuginone (3)
∆
Versatile building block
Access to both enantiomers
Facile route to biologically
interesting material
∆
N
N
3
N
O
3
N
O
R
Br
2
O
HO
N
H
2
O
HO
N
H
R'
Cl
(−)-Isohalofuginone (14)
R = R' = H, (+)-Isofebrifugine (2)
R = Br, R' = Cl, (+)-Isohalofuginone (14)

ACCEPTED MANUSCRIPT
An Asymmetric Synthesis of Febrifugine, Halofuginone and their Hemiketal
Isomers
Shaun Smullen and Paul Evans*
Centre for Synthesis and Chemical Biology, School of Chemistry, University College Dublin, Dublin 4,
Ireland. paul.evans@ucd.ie [ORCID: 0000-0002-0584-876X]
ABSTRACT
A new synthesis of both enantiomers of naturally occurring febrifugine (1) and its analogue
halofuginone (3) are reported. This robust route relies on an enzymatic kinetic resolution (EKR)-
cross metathesis (CM) sequence, performed on allylic alcohol 8. A diastereoselective Lewis acid-
mediated cyclisation of resultant enone 6 affords piperidine 12. In relation to its enantiomeric
excess, values of 87 and 88% e.e were obtained for both enantiomers of 12 and for (–)-12 this was
increased to 98% e.e by conducting the resolution process twice. A bromination (featuring an in situ
temporary alcohol protection), alkylation and amino-deprotection sequence finally afforded the
target compounds (+)- and (–)-1, and (+)- and (–)-3. Studies demonstrate that, as their ammonium
salts, the compounds are stereochemically stable. However, as their free-bases, particularly in
chloroform, C-2 isomerisation occurs. This isomerisation has been taken advantage of synthetically
to provide enantioenriched (+)-isofebrifugine (2) and both enantiomers of isohalofuginone (14).
1. INTRODUCTION
The natural product febrifugine [(+)-1] has been of interest from both a structural and biological
perspective since the first reports of its isolation in the 1940s.1 In the ensuing years this compound
has proved to be a popular synthetic target,2 however, confusion regarding the precise structure of 1
remained until Kobayashi’s synthesis in 1999.3 This confusion primarily arose due to febrifugine’s (1)
co-occurrence with (and isomerisation to) its isomer, isofebrifugine (2) (Scheme 1).
1

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bromination/
alkylation
N
HO
R
N
O
N
O
R
N
R'
N
H
HO
O
epimerisation
N
H
R'
O
cyclisation
R = R' = H, Isofebrifugine (2)
R = R' = H, Febrifugine (1)
R = Br, R' = Cl, Halofuginone (3)
PhO2S HN
Ref. 10
Cbz
R
N
O
HO
7
+
NH
R'
HN
Cbz
HO
O
6
4: R = R' = H
5: R = Br, R' = Cl
commercially available
This
work
HN
Cbz
8
Scheme 1. The structures of febrifugine (1), isofebrifugine (2), the synthetic analogue halofuginone
(3) and their retrosynthesis.
Despite promising anti-parasitic activity, 1 failed to translate into a medicine due to associated
toxicity.1,4 Efforts aimed at improving febrifugine’s activity/toxicity profile led to the synthesis of
various analogues, including halofuginone (3), in which the metabolically vulnerable protons in the
quinazolinone ring have been replaced with a bromine and chlorine atom respectively.4
Racemic halofuginone (3) has been used in veterinary medicine to combat intestinal protozoal
infections in livestock and it was during this usage that the mammalian biological properties of 3
were first noticed.5 Its ability to block protein biosynthesis has been studied.6 Halofuginone has
been the subject of human trials,7 most recently in a study focused on evaluating its ability to
improve the prognosis of patients suffering from Duchenne muscular dystrophy.8 Rather remarkably
these studies have only been conducted on racemic material and reports of enantiomer specific
biological activity of 3 are scarce.9
In 2010 we reported an enantioselective synthesis of both enantiomers of 1 and 3.10 As shown in
Scheme 1, the final part of this synthesis was based on Harayama and Takeuchi’s work,2e relying on a
diastereoselective N-conjugate addition of γ-hydroxy enone 6, followed by bromination and
alkylation of the resultant piperidine.
Compound 6 was prepared via the asymmetric
dihydroxylation of vinyl sulfone 7. Although this route proved reasonably efficient it suffered from
2

ACCEPTED MANUSCRIPT
being somewhat lengthy and the final compounds were obtained in moderately good enantiomeric
excess (85% to 90% e.e). Based on our current interest in the biological activities associated with
this compound class, particularly enantioenriched/pure 3, a revised and shorter route for the
synthesis of this class of compound was designed. Specifically this targets key building block enone
6, which would serve as a versatile building block to access material of both enantiomeric series. In
turn we envisaged that compound 6 could be obtained from the cross metathesis reaction11 of
optically active allylic alcohol 8.
2. RESULTS AND DISCUSSION
Racemic allylic alcohol 8 was prepared in five steps from pyrrolidinone 9 in 56% overall yield
(Scheme 2). Boc protection followed by subsequent Grignard addition with vinylmagnesium chloride
afforded enone 10 in good yield.12 Luche reduction of 10 gave the allylic alcohol which was finally
Cbz-protected providing (±)-8. In relation to this sequence, the direct use of Cbz-protected
pyrrolidinone proved problematic due to side-products resulting from unselective addition of the
Grignard reagent.
(1) Boc2O, DMAP,
MeCN, 0 °C to rt
(1) NaBH4, CeCl3·7H2O
MeOH, 0 °C, 91%
O
O
N
H
9
(2) Vinylmagnesium
chloride, THF, -78 °C,
83% (2 steps)
HN
Boc
(2) TFA, DCM, 0 °C
(3) CbzCl, Et3N, DCM,
0 °C to rt, 75% (2 steps)
10
CAL-B, vinyl acetate,
i-Pr2O, rt, 2.5 h
S
R
RO
HO
HO
+
HN
Cbz
HN
Cbz
HN
Cbz
( )-8
(±)-8
( )-11: R = Ac
(+)-8: R = H
K2CO3, MeOH, rt
CALB
Scheme 2. The synthesis of racemic allylic alcohol 8 and its enzymatic kinetic resolution.
With racemic alcohol 8 in hand its resolution was considered. Allylic alcohol 8 was resolved into (–)-
8 and (–)-11 using a commercially available lipase.13 The faster reacting S-enantiomer of 8 was
isolated as its acetate ester 11 in 46% yield and the more polar (–)-8 was isolated in 47% yield.
Hydrolysis of (–)-11 gave (+)-8 in 88% yield (which could be subjected to a second iteration of
3

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resolution in order to increase e.e). This process was purposely performed to access both
enantiomers of 8, ultimately targeting both enantiomers of halofuginone (3) for biological
evaluation.
The next step in the sequence was the cross metathesis of the allylic alcohol 8 with methyl vinyl
ketone (MVK). Although these two olefins are classed by Grubbs as being in the same reactivity
group11 (which would generally lead to non-selective cross-metathesis) reactions between allylic
alcohols and α,β-unsaturated carbonyl compounds have been reported.14 Thus, employing standard
reaction conditions, (+)-8 was smoothly converted into (+)-6 in the presence of excess MVK.
Subsequently, enone (+)-6 was converted into (–)-piperidine 12 using the reported Lewis acidic,
diastereoselective cyclisation (Scheme 3).10 Similarly, (–)-8 was converted into (+)-12 (not shown,
61% yield (2 steps), 88% e.e). Yields for both steps were high and with the enantiomeric forms of
piperidine 12 in hand their enantiomeric excess, set during the enzymatic kinetic resolution, was
determined by chiral HPLC.
Grubbs II
(2.5 mol%), MVK,
HO
HO
HO
BF3·OEt2
O
HN
Cbz
O
N
Cbz
DCM, rt, 88-91%
HN
Cbz
MeCN, rt, 69-78%
diastereoselective
cyclisation
( )-12
(+)-8
(+)-6
(87%/98% e.e)
N
(1) TMSOTf, DIPEA,
DCM, 0 °C, 20 min;
HO
N
O
O
N
O
N
(2) NBS, 0 °C to rt,
20 min;
(3) 4, K2CO3, DMF, rt;
(4) H2, Pd/C, MeOH, rt
N
H
(R)
CDCl3,
O
HO
N
H
∆, 24 h
(S)
(+)-1 (60%)
(+)-2 (89%)
Scheme 3. The cross metathesis reaction of 8 and the formation of optically active piperidine 12.
Next, based on the robust synthesis of both enantiomers of 12, their conversion into febrifugine and
halofuginone was considered. In our10 previous synthesis the secondary alcohol in 12 was protected
prior to functionalisation and subsequent alkylation of the methyl ketone. Apart from adding two
steps to the overall synthesis, the conversion of the secondary alcohol group into its O-benzyl ether
was low yielding. Since the methyl activation process employs TMSOTf we reasoned that it was
probably unnecessary to separately protect the secondary alcohol and that in an excess of the
4

ACCEPTED MANUSCRIPT
silylating reagent the hydroxy group will be temporarily masked as a silyl ether. In the event, this
proved to be feasible and with three equivalents of TMSOTf piperidine (–)-12 was converted into the
corresponding silyl enol ether which then underwent bromination with NBS. Following work-up the
crude bromide was directly alkylated with commercially available 3H-quinazolinone 4. Finally, the
Cbz-group was removed to afford (+)-1 as its free-base in good yield (60%) over the four steps. In an
identical fashion the use of (+)-12 gave unnatural (–)-1 (68% yield, not shown).
It has been reported1,15 that febrifugine (1) undergoes gradual isomerisation into isofebrifugine (2)
under several conditions and during characterisation of 1 we found that this process appears to be
particularly facile in CDCl3. Based upon this, (+)-1 was converted into (+)-2 on heating to reflux in
chloroform. Over the course of 24 h this transformation reached completion and good yields of (+)-
2 were obtained. The stereochemistry of the hemiketal stereogenic center is not unequivocally
known, however, NMR spectra clearly demonstrate, that in CDCl3, one diastereomer of 2 is present.
On the basis of NOE measurements it has been suggested that the stereochemistry of this center is
as shown (Scheme 3) and that a hydrogen-bond stabilizes this particular configuration.15
The same sequence of reactions was then performed using (–)- and (+)-12 and halogenated
quinazolinone 5.16 Thus both (–)- and (+)-13 could be accessed in good yields over three steps
(Scheme 4). Thereafter, the Cbz group in 13 was removed with HBr in AcOH, which afforded both
(+)- and (–)-3, as their di-HBr salts. The salts could be characterised and stored as such, or liberated
as their free-bases. In relation to this, when stored as salts no isomerisation into isohalofuginone
was detected (see ESI section for more detail).17 However, as described for febrifugine, both
enantiomers of halofuginone (3) could be converted into isohalofuginone (14) on heating the free-
base in chloroform (Scheme 4). As observed for (+)-2, in CDCl3 only one diastereomeric form of (+)-
and (–)-14 was detected.
5

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(1) TMSOTf, DIPEA,
DCM, 0 °C, 20 min;
N
HO
Br
Cl
N
HO
Br
N
HO
O
N
O
O
O
O
N
N
Br
(2) NBS, 0 °C to rt,
20 min;
(3) 5, K2CO3, DMF,
rt, 18 h
K2CO3
CDCl3,
N
R
Cl
N
H
N
H
N
Cbz
HO
∆,15 h
O
O
Cl
(+)-14 (63%)
( )-12
(87% e.e)
R = Cbz, ( )-13 (64%)
R = H, (+)-3·2HBr (60%)
(+)-3 (76%)
HBr, AcOH, rt
(1) TMSOTf, DIPEA,
DCM, 0 °C, 20 min;
N
HO
O
HO
Br
Cl
N
HO
Br
Cl
N
N
O
N
O
O
O
N
Br
K2CO3
N
(2) NBS, 0 °C to rt,
20 min;
(3) 5, K2CO3, DMF,
rt, 18 h
CDCl3,
N
Cbz
N
H
N
H
HO
R
∆,15 h
O
O
Cl
( )-14 (68%)
(+)-12
(88% e.e)
R = Cbz, (+)-13 (64%)
R = H, ( )-3·2HBr (55%)
HBr, AcOH, rt
(−)-3 (93%)
Scheme 4. The activation-alkylation of piperidine 12 in the synthesis of (+)- and (–)-halofuginone 3.
The isomerisation of (+)- and (–)-halofuginone 3 into (+)- and (–)-isohalofuginone 14.
3. CONCLUSION
In summary, a straight-forward 13 step synthesis of the quinazolinone-containing alkaloid (+)-and (–
)-febrifugine 1 has been developed. The route relies on an enzymatic kinetic resolution and a cross
metathesis reaction. Overall yields of 11% for both enantiomers from pyrrolidinone 9 were
observed. The enantiomeric excess of the final compound was 87% (improved to 98% e.e if the
initially resolved material was re-subjected to the resolution process) and 88% respectively. In a
similar manner both enantiomers of halofuginone (3) were obtained. Access to optically active 3 is
relevant based on the current biological/clinical studies using (±)-3. The C-2 isomerisation of
febrifugine and halofuginone into their hemiketal isomers was also studied and the observation that
the free-secondary amines undergo gradual isomerisation in CDCl3 was taken advantage of
synthetically. In relation to the latter (14), this study constitutes the first total synthesis of these
compounds.
4. EXPERIMENTAL SECTION
4.1. General Directions
Starting materials were supplied from commercial sources and used without further purification
unless otherwise stated. All commercially available solvents were used as supplied unless otherwise
6

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stated. All “dry” solvents were dried and distilled by standard procedures or were processed through
a Grubbs-type still. Oxygen-free nitrogen was used without further drying. Infrared spectroscopy was
performed on a FT-IR spectrometer. Routine electrospray mass spectra and high-resolution mass
spectra were performed by electrospray ionization (ESI). Chiral HPLC was performed on a Chiralpak
IA column. The NMR spectra were recorded at 25 °C on 300, 400, 500 MHz spectrometers as
indicated. The peak assignments given were confirmed by 2D experiments (1H-13C HSQC). TLC (thin
layer chromatography) was performed on 60F254 aluminium plates with realisation by UV irradiation.
Flash column chromatography was performed with silica, particle size 40 – 63 μm.
ο
4.2. tert-Butyl pyrrolidinone-1-carboxylate:18 At 0 C, to a stirred solution of pyrrolidinone 9 (3.83
mL, 50.0 mmol, 1.0 eq.) in MeCN (100 mL) was added Boc2O (13.09 g, 60.0 mmol, 1.2 eq.) and DMAP
(0.61 g, 5.0 mmol, 10 mol%). The reaction mixture was warmed to room temperature and stirred for
4 h. The reaction mixture was concentrated in vacuo, dissolved in EtOAc (200 mL), washed with
0.025 M aq. HCl (200 mL), and back extracted with EtOAc (200 mL). The combined organic layers
were washed with brine (400 mL), dried over MgSO4, filtered, and concentrated in vacuo to afford
the crude product which was purified by flash column chromatography (c-Hex/EtOAc; 1:1) affording
1
the title compound (8.81 g, 95%) as an orange oil. Rf = 0.3 (c-Hex/EtOAc; 1:1); H NMR (CDCl3, 300
MHz): δ 1.53 (s, 9H), 2.00 (quin, J = 8.0 Hz, 2H), 2.51 (t, J = 8.0 Hz, 2H), 3.75 (t, J = 8.0 Hz, 2H).
ο
4.3. tert-Butyl (4-oxohex-5-en-1-yl)carbamate 10: Under N2 at −78 C, to a stirred solution of tert-
butyl pyrrolidinone-1-carboxylate (8.81 g, 47.6 mmol, 1.0 eq.) in dry THF (250 mL) was added 1.6 M
vinylmagnesium chloride in THF (30 mL, 48.0 mmol, 1.0 eq.) in a dropwise fashion. The reaction
mixture was stirred at −78 οC for 0.5 h. Another aliquot of 1.6 M vinylmagnesium chloride in THF (30
mL, 48.0 mmol, 1.0 eq.) was added in a dropwise fashion and the reaction mixture was stirred at −78
οC for a further 0.5 h. The reaction mixture was quenched with MeOH/AcOH (40 mL, 1:1), diluted
with Et2O (250 mL), and washed with H2O (500 mL) and brine (500 mL). The organic layer was dried
over MgSO4, filtered, and concentrated in vacuo to afford the crude product which was purified by
7

ACCEPTED MANUSCRIPT
flash column chromatography (c-Hex/EtOAc; 2:1) affording 10 (8.83 g, 87%) as a yellow solid after
ο
storage in a freezer. Rf = 0.3 (c-Hex/EtOAc; 2:1); Mp = 35 – 38 C; IR (neat): 3373, 3001, 2980, 2950,
2930, 1710, 1670, 1530, 1365, 1328, 1271, 1248, 1163 cm-1; HRMS (ESI): calcd. for [C11H19NO3 + Na]+
236.1263, found 236.1257; 1H NMR (CDCl3, 400 MHz): δ 1.44 (s, 9H), 1.82 (quin, J = 7.0 Hz, 2H), 2.65
(t, J = 7.0 Hz, 2H), 3.16 (q, J = 7.0 Hz, 2H), 4.72 (br. s, 1H), 5.84 (d, J = 10.5 Hz, 1H), 6.23 (d, J = 17.5 Hz,
1H), 6.36 (dd, J = 17.5, 10.5 Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 24.1 (CH2), 28.4 (CH3), 36.7
(CH2), 40.0 (CH2), 79.1 (C), 128.2 (CH2), 136.4 (CH), 156.0 (C), 200.2 (C) ppm.
ο
4.4. tert-Butyl (4-hydroxyhex-5-en-1-yl)carbamate:19 At 0 C, to a stirred solution of NaBH4 (0.87 g,
23.0 mmol, 2.5 eq.) and CeCl3.7H2O (3.76 g, 10.1 mmol, 1.1 eq.) in MeOH (50 mL) was added 10 (1.96
g, 9.2 mmol, 1.0 eq.) in MeOH (50 mL) in a dropwise fashion. The reaction mixture was stirred at 0 οC
for 25 min. The reaction mixture was monitored by TLC, an additional aliquot of NaBH4 (0.35 g, 9.2
ο
mmol, 1.0 eq.) was added and the reaction mixture was stirred at 0 C for a further 10 min. The
reaction mixture was quenched with 0.2 M aq. HCl (120 mL) and extracted with EtOAc (3 x 200 mL).
The organic layers were combined, dried over MgSO4, filtered, and concentrated in vacuo to afford
the crude product which was purified by flash column chromatography (c-Hex/EtOAc; 2:1) affording
the title compound (1.80 g, 91%) as a colourless oil. Rf = 0.3 (c-Hex/EtOAc; 1:1); IR (neat): 3340. 3079,
3005, 2977, 2869, 1684, 1516, 1453, 1366, 1250, 1166, 990 cm-1; HRMS (ESI): calcd. for [C11H21NO3 +
1
Na]+ 238.1419, found 238.1420; H NMR (CDCl3, 400 MHz): δ 1.44 (s, 9H), 1.50 – 1.66 (m, 4H), 1.75
(br. s, 1H), 3.06 – 3.24 (m, 2H), 4.13 (q, J = 6.0 Hz, 1H), 4.61 (br. s, 1H), 5.11 (d, J = 10.5 Hz, 1H), 5.23
(d, J = 17.0 Hz, 1H), 5.86 (ddd, J = 17.0, 10.5, 6.0 Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ 26.0
(CH2), 28.4 (CH3), 33.9 (CH2), 40.3 (CH2), 72.8 (CH), 79.1 (C), 114.8 (CH2), 141.0 (CH), 156.0 (C) ppm.
4.5. (±)-Benzyl (4-hydroxyhex-5-en-1-yl)carbamate 8:20 At 0 οC, to a stirred solution of tert-butyl (4-
hydroxyhex-5-en-1-yl)carbamate (0.380 g, 1.77 mmol, 1.0 eq.) in DCM (7 mL) was added TFA (2.7
ο
mL, 35.3 mmol, 20.0 eq.) in a dropwise fashion. The reaction mixture was stirred at 0 C for 45 min.
The reaction mixture was concentrated in vacuo. DCM (10 mL) was added and the solvent removed
8

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in vacuo again. This process was repeated two more times to afford the ammonium salt. At 0 οC, to a
stirred solution of the ammonium salt in DCM (20 mL) was added Et3N (1.25 mL, 8.97 mmol, 5.0 eq.)
in a dropwise fashion and then CbzCl (0.30 ml, 2.10 mmol, 1.2 eq.). The reaction mixture was
warmed to room temperature and stirred for 18 h. The reaction mixture was quenched with sat. aq.
NH4Cl (20 mL), separated, and washed with brine (20 mL). The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo to afford the crude product which was purified by flash column
chromatography (c-Hex/EtOAc; 3:2) affording 8 (0.330 g, 75%) as a colourless oil. Rf = 0.3 (c-
Hex/EtOAc; 1:1); IR (neat): 3410, 3328, 2940, 2869, 1693, 1525, 1454, 1412, 1371, 1251, 1135, 1020
cm-1; HRMS (ESI): calcd. for [C14H19NO3 + Na]+ 272.1263, found 272.1250; 1H NMR (CDCl3, 400 MHz):
δ 1.49 – 1.78 (m, 4H), 3.24 (q, J = 6.5 Hz, 2H), 4.13 (q, J = 6.5 Hz, 1H), 4.87 (br. s, 1H), 5.05 – 5.15 (m,
3H), 5.22 (d, J = 17.0 Hz, 1H), 5.85 (ddd, J = 17.0, 10.5, 6.5 Hz, 1H), 7.28 – 7.42 (m, 5H) ppm; 13C NMR
(CDCl3, 100 MHz): δ 25.9 (CH2), 33.8 (CH2), 40.9 (CH2), 66.6 (CH2), 72.8 (CH), 114.9 (CH2), 128.1 (CH),
128.1 (CH), 128.5 (CH), 136.6 (C), 140.9 (CH), 156.5 (C) ppm.
4.6. (−)-(S)-Benzyl (4-acetoxyhex-5-en-1-yl)carbamate 11 and (−)-(R)-benzyl (4-hydroxyhex-5-en-1-
yl)carbamate 8: To a stirred solution of (±)-8 (0.469 g, 1.88 mmol, 1.0 eq.) in i-Pr2O (20 mL) with 3 Å
molecular sieves (0.2 g) was added vinyl acetate (0.87 mL, 9.44 mmol, 5.0 eq.) and CAL-B (0.470 g).
1
The reaction mixture was monitored by H NMR spectroscopy and stirred gently for 2.5 h. The
reaction mixture was filtered, washed with Et2O (2 x 20 mL) and EtOAc (2 x 20 mL), and concentrated
in vacuo to afford the crude product which was purified by flash column chromatography (c-
20
Hex/EtOAc; 2:1) affording (−)-11 (0.257 g, 47%) as a colourless oil. Rf = 0.5 (c-Hex/EtOAc; 1:1); [α]D
= −2.8 (c = 1.00, CHCl3); IR (neat): 3346, 2945, 2871, 1741, 1698, 1526, 1454, 1371, 1231, 1134, 1078,
1
1018 cm-1; HRMS (ESI): calcd. for [C16H21NO4 + Na]+ 314.1368, found 314.1359; H NMR (CDCl3, 500
MHz): δ 1.49 – 1.74 (m, 4H), 2.07 (s, 3H), 3.22 (q, J = 6.5 Hz, 2H), 4.79 (br. s, 1H), 5.10 (s, 2H), 5.17 (d,
J = 10.5 Hz, 1H), 5.21 – 5.28 (m, 2H), 5.76 (ddd, J = 17.0, 10.5, 6.5 Hz, 1H), 7.30 – 7.40 (m, 5H) ppm;
13C NMR (CDCl3, 125 MHz): δ 21.2 (CH3), 25.7 (CH2), 31.3 (CH2), 40.8 (CH2), 66.7 (CH2), 74.3 (CH),
9

ACCEPTED MANUSCRIPT
117.0 (CH2), 128.1 (2 x CH), 128.5 (CH), 136.1 (CH), 136.6 (C), 156.3 (C), 170.3 (C) ppm. Further
20
elution gave (−)-8 (0.216 g, 46%) as a colourless oil. [α]D = −3.2 (c = 1.00, CHCl3); with additional
data as above for (±)-8.
4.7. (+)-(S)-Benzyl (4-hydroxyhex-5-en-1-yl)carbamate 8: To a stirred solution of (−)-11 (0.237 g,
0.81 mmol, 1.0 eq.) in MeOH (10 mL) was added K2CO3 (0.169 g, 1.22 mmol, 1.5 eq.). The reaction
mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo,
dissolved in DCM (10 mL), washed with H2O (10 mL) and back extracted with DCM (2 x 10 mL). The
combined organic layers were washed with brine (30 mL), dried over MgSO4, filtered, and
concentrated in vacuo to afford the crude product which was purified by flash column
chromatography (c-Hex/EtOAc; 3:2) affording (+)-8 (0.179 g, 88%) as a colourless oil. [α]D20 = +3.6 (c
= 1.00, CHCl3); with additional data as above for (±)-8.
4.7.1. Second iteration of enzymatic kinetic resolution: (+)-(S)-Benzyl (4-hydroxyhex-5-en-1-
yl)carbamate 8: In an identical procedure to that described above for the resolution of (±)-8 and
deacetylation of (−)-11, enantioenriched (+)-8 (0.146 g, 0.59 mmol, 1.0 eq.), obtained after one
iteration of enzymatic kinetic resolution (82% e.e, determined by HPLC analysis after conversion to
(−)-12), was treated with vinyl acetate (0.27 mL, 2.93 mmol, 5.0 eq.) and CAL-B (0.146 g) in i-Pr2O (5
mL) with 3 Å molecular sieves (0.050 g) for 1.5 h affording, after purification by flash column
chromatography (c-Hex/EtOAc; 2:1), (−)-11 (0.127 g, 74%) as a clear oil. (−)-11 (0.111 g, 0.38 mmol,
1.0 eq.) was then treated with K2CO3 (0.079 g, 0.57 mmol, 1.5 eq.) in MeOH (5 mL) affording, after
purification by flash column chromatography (c-Hex/EtOAc; 3:2), (+)-8 (0.079 g, 83%, 98% e.e,
determined by HPLC analysis after conversion to (−)-12) as a clear oil. Data as above for (−)-11 and
(+)-8.
4.8. (−)-(R,E)-Benzyl (4-hydroxy-7-oxooct-5-en-1-yl)carbamate 6: Under N2, to a stirred solution of
(−)-8 (0.196 g, 0.79 mmol, 1.0 eq.) in dry, N2 purged DCM (3 mL) was added methyl vinyl ketone
(0.33 mL, 3.96 mmol, 5.0 eq.) and Grubbs’ II catalyst (0.017 g, 0.02 mmol, 2.5 mol%). The reaction
10

ACCEPTED MANUSCRIPT
mixture was stirred at room temperature for 18 h before concentration in vacuo and purification by
flash column chromatography (c-Hex/EtOAc; 1:2) which afforded (−)-6 (0.192 g, 84%) as a light
20
brown oil. Rf = 0.2 (c-Hex/EtOAc; 1:2); [α]D = −9.3 (c = 1.00, CHCl3); IR (neat): 3336, 2929, 2871,
1693, 1673, 1631, 1529, 1454, 1337, 1252, 1180, 1021 cm-1; HRMS (ESI): calcd. for [C16H21NO4 + Na]+
314.1368, found 314.1375; 1H NMR (CDCl3, 400 MHz): δ 1.53 – 1.76 (m, 4H), 2.27 (s, 3H), 2.21 – 2.36
(m, 1H), 3.17 – 3.31 (m, 2H), 4.11-4.41 (m, 1H), 4.89 (br. s, 1H), 5.09 (s, 2H), 6.27 (d, J = 16.0 Hz, 1H),
6.73 (dd, J = 16.0, 5.0 Hz, 1H), 7.29 – 7.39 (m, 5H) ppm; 13C NMR (CDCl3, 100 MHz): δ 26.0 (CH2), 27.5
(CH3), 33.2 (CH2), 40.6 (CH2), 66.8 (CH2), 70.8 (CH), 128.1 (CH), 128.2 (CH), 128.5 (CH), 129.2 (CH),
136.5 (C), 148.4 (CH), 156.6 (C), 198.4 (C) ppm.
4.9. (+)-(S,E)-Benzyl (4-hydroxy-7-oxooct-5-en-1-yl)carbamate 6:2e In an identical procedure to that
described above for (−)-6, (+)-8 (0.160 g, 0.64 mmol, 1.0 eq.) was treated with methyl vinyl ketone
(0.27 mL, 3.24 mmol, 5.0 eq.) and Grubbs’ II catalyst (0.014 g, 0.02 mmol, 2.5 mol%) in dry, N2
purged DCM (3 mL) affording, after purification by flash column chromatography (c-Hex/EtOAc; 1:2),
(+)-6 (0.170 g, 91%) as a light brown oil. [α]D20 = +8.3 (c = 1.00, CHCl3); {lit.,2e [α]D19 = +12.3 (c = 1.00,
EtOH)}; with additional data as above for (−)-6.
4.10. (+)-(2S,3R)-Benzyl 3-hydroxy-2-(2-oxopropyl)piperidine-1-carboxylate 12:10 To a stirred
solution of (−)-6 (0.179 g, 0.61 mmol, 1.0 eq.) in MeCN (5 mL) was added BF3∙OEt2 (39 μL, 0.031
mmol, 50 mol%) in MeCN (1 mL). The reaction mixture was stirred at room temperature for 10 min.
The reaction mixture was diluted with DCM (5 mL), quenched with sat. aq. NaCHO3 (10 mL),
separated, and washed with brine (10 mL). The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo to afford the crude product which was purified by flash column
chromatography (c-Hex/EtOAc; 1:2) affording (+)-12 (0.131 g, 73%) as a light brown oil. Rf = 0.1 (c-
20
20
Hex/EtOAc; 1:2); [α]D = +19.5 (c = 1.00, CHCl3); {lit.,10 [α]D = +22.8 (c = 1.00, CHCl3)}; IR (neat):
3438, 2941, 2871, 1673, 1423, 1349, 1191, 1166, 1125, 1077, 1044, 1029 cm-1; HRMS (ESI): calcd. for
[C16H21NO4 + Na]+ 314.1368, found 314.1365; 1H NMR (CDCl3, 400 MHz): δ 1.36 – 1.49 (m, 1H), 1.60 –
11

ACCEPTED MANUSCRIPT
1.99 (m, 3H), 2.04 – 2.26 (m, 3H), 2.65 (d, J = 7.0 Hz, 2H), 2.87 (br. t, J = 12.5 Hz, 1H), 3.76 – 3.86 (m,
1H), 4.07 (br. d, J = 12.5 Hz, 1H), 4.73 (br. t, J = 7.0 Hz, 1H), 5.13 (s, 2H), 7.28 – 7.40 (m, 5H) ppm; 13
C
NMR (CDCl3, 100 MHz): δ 18.9 (CH2), 25.9 (CH2), 30.0 (CH3), 39.6 (CH2), 43.7 (CH2), 54.1 (CH), 67.0
(CH), 67.3 (CH2), 127.8 (CH), 128.0 (CH), 128.5 (CH), 136.6 (C), 156.3 (C), 206.0 (C) ppm; HPLC analysis
(Chiralpak IA), heptane/EtOH; 70:30 (1 mL/min): (+)-12 = 10.2 min, (−)-12 = 13.8 min; 88% e.e.
4.11. (−)-(2R,3S)-Benzyl 3-hydroxy-2-(2-oxopropyl)piperidine-1-carboxylate 12:2e,10 In an identical
procedure to that described above for (−)-12, (+)-6 (0.158 g, 0.54 mmol, 1.0 eq.) was treated with
BF3∙OEt2 (34 μL, 0.27 mmol, 50 mol%) in MeCN (6 mL) affording, after purification by flash column
chromatography (c-Hex/EtOAc; 1:2), (−)-12 (0.124 g, 78%) as a light brown oil. [α]D20 = -17.6 (c = 1.00,
20
CHCl3); {lit.,10 [α]D = -18.4 (c = 0.50, CHCl3)}; HPLC analysis (Chiralpak IA), heptane/EtOH; 70:30 (1
mL/min): (+)-12 = 9.8 min, (−)-12 = 12.7 min; 87% e.e; with additional data as above for (+)-12.
4.12.
(−)-(2R,3S)-Benzyl
3-hydroxy-2-(2-oxo-3-(4-oxoquinazolin-3(4H)-yl)propyl)piperidine-1-
ο
carboxylate (N-Cbz-protected febrifugine): Under N2 at 0 C, to a stirred solution of (−)-12 (81 mg,
0.28 mmol, 1.0 eq.) in dry DCM (5 mL) was added DIPEA (0.15 mL, 0.89 mmol, 3.2 eq.) and TMSOTf
ο
(0.16 mL, 0.89 mmol, 3.2 eq.). The reaction mixture was stirred at 0 C for 20 min. To the reaction
mixture was added NBS (69 mg, 0.39 mmol, 1.4 eq.). The reaction mixture was warmed to room
temperature and stirred for a further 20 min. The reaction mixture was washed with H2O (5 mL) and
brine (5 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to afford
the crude α-brominated intermediate. To a stirred solution of the crude α-brominated intermediate
in DMF (5 mL) was added 4 (41 mg, 0.28 mmol, 1.0 eq.) and K2CO3 (38 mg, 0.28 mmol, 1.0 eq.). The
reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with
EtOAc (10 mL), washed with H2O (10 mL), and back extracted with EtOAc (10 mL). The combined
organic layers were washed with brine (20 mL), dried over MgSO4, filtered, and concentrated in
vacuo to afford the crude product which was purified by flash column chromatography (EtOAc)
affording (−)-Cbz-protected febrifugine (75 mg, 62%) as an off-white solid. Rf = 0.1 (EtOAc); Mp = 253
12

ACCEPTED MANUSCRIPT
ο
20
– 257 C (decomp.); [α]D = -26.9 (c = 1.00, CHCl3); IR (neat): 3430, 2928, 2858, 1728, 1668, 1610,
1472, 1423, 1360, 1321, 1260, 986 cm-1; HRMS (ESI): calcd. for [C24H25N3O5 + H]+ 436.1872, found
436.1878; 1H NMR (CDCl3, 400 MHz): δ 1.38 – 1.48 (m, 1H), 1.63 – 1.83 (m, 2H), 1.83 – 1.99 (m, 1H),
2.80 (d, J = 7.5 Hz, 2H), 2.98 (br. t, J = 13.5 Hz, 1H), 3.12 (br. s, 1H), 3.83 – 3.89 (m, 1H), 4.01 (br. d, J =
13.5 Hz, 1H), 4.66 – 5.01 (m, 3H), 5.10 (s, 2H), 7.21 – 7.35 (m, 5H), 7.47 (ddd, J = 8.0, 7.0, 1.5 Hz, 1H),
7.66 – 7.79 (m, 2H), 7.90 (br. s, 1H), 8.23 (dd, J = 8.0, 1.5 Hz, 1H) ppm; 13C NMR (CDCl3, 100 MHz): δ
19.0 (CH2), 26.1 (CH2), 39.8 (CH2), 40.8 (CH2), 53.9 (CH2), 54.8 (CH), 67.0 (CH), 67.5 (CH2), 121.7 (C),
126.7 (CH), 127.3 (CH), 127.5 (CH), 127.8 (CH), 128.1 (CH), 128.5 (CH), 134.5 (CH), 136.4 (C), 146.7
(CH), 148.1 (C), 156.6 (C), 160.9 (C), 200.3 (C) ppm.
4.13.
(+)-(2S,3R)-Benzyl
3-hydroxy-2-(2-oxo-3-(4-oxoquinazolin-3(4H)-yl)propyl)piperidine-1-
carboxylate (N-Cbz-protected febrifugine): In an identical procedure to that described above (+)-12
(285 mg, 0.98 mmol, 1.0 eq.) was treated with DIPEA (0.55 mL, 3.13 mmol, 3.2 eq.), TMSOTf (0.57
mL, 3.13 mmol, 3.2 eq.) and then NBS (244 mg, 1.37 mmol, 1.4 eq.) in dry DCM (10 mL) followed by
5 (143 mg, 0.98 mmol, 1.0 eq.) and K2CO3 (143 mg, 0.98 mmol, 1.0 eq.) in DMF (10 mL) affording,
after purification by flash column chromatography (EtOAc), (+)-Cbz-protected febrifugine (307 mg,
72%) as an off-white solid. [α]D20 = +28.0 (c = 1.00, CHCl3); with additional data as above for (−)-Cbz-
protected febrifugine.
4.14. (+)-Febrifugine 1:3 To a stirred solution of (−)-Cbz-protected febrifugine (46 mg, 0.11 mmol, 1.0
eq.) in MeOH (2 mL) was added 10% wt. Pd/C (22 mg, 0.02 mmol, 20 mol%). The reaction mixture
was stirred at room temperature under a H2 atmosphere for 1.5 h. The reaction mixture was filtered
through a pad of celite, washed with EtOAc (10 mL), and concentrated in vacuo to afford (+)-1 (31
mg, 97%) as a white solid. Mp = 133 – 136 οC; {lit.,3 Mp = 138 – 140 οC, (–)-1}; [α]D20 = +14.0 (c = 0.50,
27
EtOH); {lit.,3 [α]D = +26.6 (c = 0.1, EtOH)}; IR (neat): 2927, 2848, 1726, 1668, 1610, 1562, 1472,
1449, 1398, 1363, 1141, 1001 cm-1; HRMS (ESI): calcd. for [C16H19N3O3 + H]+ 302.1505, found
1
302.1504; H NMR ((CD3)2SO, 500 MHz): δ 1.14 – 1.43 (m, 2H), 1.52 – 1.62 (m, 1H), 1.84 – 1.97 (m,
13

ACCEPTED MANUSCRIPT
1H), 2.33 – 2.48 (m, 2H), 2.61 – 2.72 (m, 1H), 2.74 – 2.85 (m, 1H), 2.94 – 3.05 (m, 2H), 4.70 – 4.87 (m,
1H), 4.93 – 5.05 (m, 2H), 7.56 (t, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 8.14
(d, J = 8.0 Hz, 1H), 8.18 (s, 1H) ppm; 13C NMR ((CD3)2SO, 125 MHz): δ 26.3 (CH2), 34.7 (CH2), 44.2
(CH2), 46.1 (CH2), 55.1 (CH2), 60.6 (CH), 71.2 (CH), 121.8 (C), 126.5 (CH), 127.6 (CH), 127.7 (CH), 135.0
(CH), 148.4 (C), 148.5 (CH), 160.4 (C), 204.3 (C) ppm.
4.15. (−)-Febrifugine 1: In an identical procedure to that described above for (+)-1, (+)-Cbz-
protected febrifugine (151 mg, 0.35 mmol, 1.0 eq.) was treated with 10% wt. Pd/C (0.074 g, 0.07
mmol, 20 mol%) under a H2 atmosphere in MeOH (5 mL) affording, after filtration through a pad of
20
celite, (−)-1 (99 mg, 95%) as a white solid. [α]D = −8.4 (c = 0.50, EtOH); with additional data as
above for (+)-1.
4.16. (+)-Isofebrifugine 2:3 (+)-Febrifugine 1 (9.0 mg, 0.030 mmol) was stirred and heated to reflux in
CDCl3 (2 mL) for 24 h. The reaction mixture was concentrated in vacuo and purified by column
chromatography (CHCl3/MeOH; 4:1) to afford (+)-2 (8.0 mg, 89%) as an off-white solid. Rf = 0.1
ο
ο
20
(CHCl3/MeOH; 4:1); Mp = 118 – 121 C; {lit.21 Mp = 134 – 135 C, (±)-2}; [α]D = +75.6 (c = 0.50,
25
CHCl3); {lit.,3 [α]D = +129.3 (c = 0.20, CHCl3)}; IR (neat): 3307, 2927, 2854, 1668, 1610, 1565, 1473,
1432, 1366, 1094, 1058, 1012 cm-1; HRMS (ESI): calcd. for [C16H19N3O3 + H]+ 302.1505, found
302.1503; 1H NMR (CDCl3, 400 MHz): δ 1.46 – 1.63 (m, 2H), 1.76 – 1.93 (m, 2H), 2.05 – 2.19 (m, 2H),
2.47 – 2.61 (m, 1H), 2.94 – 3.06 (m, 1H), 3.26 – 3.34 (m, 1H), 3.85 – 3.93 (m, 1H), 4.16 (d, J = 14.0 Hz,
1H), 4.45 (d, J = 14.0 Hz, 1H), 7.50 (ddd, J = 8.0, 7.0, 1.5 Hz, 1H), 7.67 – 7.82 (m, 2H), 8.27 – 8.35 (m,
2H) ppm; 13C NMR (CDCl3, 100 MHz): δ 20.0 (CH2), 26.8 (CH2), 43.3 (CH2), 44.5 (CH2), 49.9 (CH2), 55.7
(CH), 77.7 (CH), 105.4 (C), 121.9 (C), 126.9 (CH), 127.0 (CH), 127.5 (CH), 134.3 (CH), 148.1 (C), 148.2
(CH), 161.4 (C) ppm.
4.17.
(+)-(2S,3R)-Benzyl
2-(3-(7-bromo-6-chloro-4-oxoquinazolin-3(4H)-yl)-2-oxopropyl)-3-
hydroxypiperidine-1-carboxylate (N-Cbz-protected halofuginone) 13: In an identical procedure to
that described above, (+)-12 (72 mg, 0.25 mmol, 1.0 eq.) was treated with DIPEA (0.14 mL, 0.79
14

ACCEPTED MANUSCRIPT
mmol, 3.2 eq.), TMSOTf (0.14 mL, 0.79 mmol, 3.2 eq.) and then NBS (62 mg, 0.35 mmol, 1.4 eq.) in
dry DCM (3.5 mL) followed by 5 (64 mg, 0.25 mmol, 1.0 eq.) and K2CO3 (34 mg, 0.25 mmol, 1.0 eq.) in
DMF (2.5 mL) affording, after purification by flash column chromatography (EtOAc), (+)-13 (87 mg,
ο
20
64%) as an off-white solid. Rf = 0.1 (EtOAc); Mp = 246 – 250 C (decomp.); [α]D = +22.7 (c = 1.00,
CHCl3); IR (neat): 3443, 2928, 2857, 1729, 1672, 1604, 1446, 1426, 1360, 1306, 1262, 1078 cm-1;
1
HRMS (ESI): calcd. for [C24H2379Br35ClN3O5 + Na]+ 570.0407, found 570.0428; H NMR (CDCl3, 400
MHz): δ 1.42 – 1.53 (m, 1H), 1.66 – 1.84 (m, 2H), 1.84 – 1.99 (m, 1H), 2.60 (br. s, 1H), 2.76 – 2.89 (m,
2H), 3.02 (br. t, J = 13.5 Hz, 1H), 3.85 – 3.91 (m, 1H), 4.01 (br. d, J = 13.5 Hz, 1H), 4.65 – 5.00 (m, 3H),
5.11 (s, 2H), 7.25 – 7.37 (m, 5H), 7.85 (br. s, 1H), 8.00 (s, 1H), 8.28 (s, 1H) ppm; 13C NMR (CDCl3, 100
MHz): δ 19.0 (CH2), 26.2 (CH2), 39.8 (CH2), 40.8 (CH2), 53.8 (CH2), 54.9 (CH), 67.2 (CH), 67.6 (CH2),
121.8 (C), 127.5 (CH), 127.7 (CH), 128.1 (CH), 128.5 (CH), 129.5 (C), 132.7 (CH), 133.6 (C), 136.2 (C),
147.1 (C), 147.8 (CH), 156.7 (C), 159.5 (C), 199.7 (C) ppm.
4.18.
(−)-(2R,3S)-Benzyl
2-(3-(7-bromo-6-chloro-4-oxoquinazolin-3(4H)-yl)-2-oxopropyl)-3-
hydroxypiperidine-1-carboxylate (N-Cbz-protected halofuginone) 13: In an identical procedure to
that described above (−)-12 (108 mg, 0.37 mmol, 1.0 eq.) was treated with DIPEA (0.21 mL, 1.19
mmol, 3.2 eq.), TMSOTf (0.21 mL, 1.19 mmol, 3.2 eq.) and then NBS (92 mg, 0.52 mmol, 1.4 eq.) in
dry DCM (5 mL) followed by 5 (96 mg, 0.37 mmol, 1.0 eq.) and K2CO3 (51 mg, 0.37 mmol, 1.0 eq.) in
DMF (4 mL) affording, after purification by flash column chromatography (EtOAc), (−)-13 (129 mg,
64%) as an off-white solid. [α]D20 = −21.6 (c = 1.00, CHCl3); with additional data as above for (+)-13.
4.19. (−)-Halofuginone 3:9,10 (+)-Cbz-protected halofuginone 13 (36 mg, 0.066 mmol) was stirred in
33% HBr in AcOH (1 mL) at room temperature for 3 h. The reaction mixture was diluted with MeOH
(1 mL), triturated with Et2O (3 x 10 mL), and dried in vacuo affording (–)-3·2HBr (21 mg, 55%) as a
ο
20
white solid. Mp = 247 – 249 C (decomp.); [α]D = −11.2 (c = 0.50, (CH3)2SO); IR (neat): 3326, 3191,
2935, 2812, 1728, 1677, 1603, 1448, 1359, 1306, 1268, 1217, 1091 cm-1; HRMS (ESI): calcd. for
1
[C16H1779Br35ClN3O3 + H]+ 414.0220, found 414.0240; H NMR ((CD3)2SO, 500 MHz): δ 1.42 – 1.50 (m,
15

ACCEPTED MANUSCRIPT
1H), 1.56 – 1.68 (m, 1H), 1.78 – 1.86 (m, 1H), 1.90 – 1.96 (m, 1H), 2.83 – 2.96 (m, 2H), 3.13 – 3.20 (m,
1H), 3.24 (dd, J = 17.5, 5.5 Hz, 1H), 3.28 – 3.36 (m, 1H), 3.46 – 3.53 (m, 1H), 5.06 (d, J = 18.0 Hz, 1H),
5.11 (d, J = 18.0 Hz, 1H), 8.19 (s, 1H), 8.23 (s, 1H), 8.28 (s, 1H), 8.54 – 8.70 (m, 2H) ppm (OH not
1
visible by H NMR spectroscopy); 13C NMR ((CD3)2SO, 125 MHz): δ 20.6 (CH2), 30.9 (CH2), 39.9 (CH2),
43.5 (CH2), 54.9 (CH2), 56.6 (CH), 67.3 (CH), 122.2 (C), 127.4 (CH), 129.0 (C), 132.3 (C), 132.9 (CH),
147.7 (C), 150.1 (CH), 159.2 (C), 201.3 (C) ppm. The salt (–)-3·2HBr was basified to pH 10 with 0.1 M
aq. K2CO3 and extracted with DCM (5 x 5 mL). The combined organic layers were washed with brine
(25 mL), dried over MgSO4, filtered, and concentrated in vacuo (without heating) to afford (−)-3 (14
ο
o
20
mg, 93%) as a white solid. Mp = 143 – 145 C; {lit.,9 Mp = 160 C (decomp.)}; [α]D = −5.4 (c = 0.50,
EtOH); {lit.,9 [α]D20 = −10 (c = 0.5, DMF)}; IR (neat): 3128, 2926, 2852, 1722, 1674, 1607, 1448, 1382,
1309, 1223, 1106, 1075, 1043, 900 cm-1; HRMS (ESI): calcd. for [C16H1779Br35ClN3O3 + H]+ 414.0220,
found 414.0212; 1H NMR ((CD3)2SO, 500 MHz): δ 1.16 – 1.42 (m, 2H), 1.52 – 1.61 (m, 1H), 1.84 – 1.95
(m, 1H), 2.32 – 2.40 (m, 1H), 2.45 (dd, J = 15.5, 9.0 Hz, 1H), 2.64 (td, J = 9.0, 4.0 Hz, 1H), 2.75 – 2.84
(m, 1H), 2.93 – 3.03 (m, 2H), 4.76 (d, J = 6.0 Hz, 1H), 4.94 – 5.06 (m, 2H), 8.16 (s, 1H), 8.22 (s, 1H),
1
8.24 (s, 1H) ppm (OH not visible by H NMR spectroscopy); 13C NMR ((CD3)2SO, 125 MHz): δ 26.3
(CH2), 34.7 (CH2), 44.1 (CH2), 46.0 (CH2), 55.3 (CH2), 60.5 (CH), 71.2 (CH), 122.2 (C), 127.4 (CH), 128.8
(C), 132.2 (C), 132.8 (CH), 147.8 (C), 150.2 (CH), 159.1 (C), 203.9 (C) ppm.
4.20. (+)-Halofuginone 3:9,10 In an identical procedure to that described above for (−)-3, (−)-13 (38
mg, 0.069 mmol) was treated with 33% HBr in AcOH (1 mL) affording, after purification as above, (+)-
3·2HBr (24 mg, 60%) as a white solid. [α]D20 = +7.3 (c = 0.50, (CH3)2SO); with additional data as above
for (−)-3·2HBr. Basification to pH 10 with 0.1 M aq. K2CO3 gave (+)-3 (13 mg, 76%) as a white solid.
[α]D20 = +5.6 (c = 0.50, EtOH); {lit.,9 [α]D25 = +9.4 (c = 0.5, DMF)}; with additional data as above for (−)-
3.
4.21. (−)-Isohalofuginone 14: In an identical procedure to that described above for (+)-2, (−)-3 (11.0
mg, 0.027 mmol) was heated to reflux in CDCl3 (1 mL) for 24 h affording, after purification by flash
16

ACCEPTED MANUSCRIPT
column chromatography (CHCl3/MeOH; 4:1), (−)-14 (7.5 mg, 68%) as an off-white solid. Rf = 0.1
ο
o
20
(CHCl3/MeOH; 4:1); Mp = 142 – 145 C; {lit.,15 Mp = 137 – 141 C, (±)-14}; [α]D = −20.2 (c = 0.50,
CHCl3); IR (neat): 3307, 2923, 2853, 1676, 1603, 1446, 1378, 1365, 1305, 1261, 1098, 1009 cm-1;
HRMS (ESI): calcd. for [C16H1779Br35ClN3O3 + H]+ 414.0220, found 414.0220; 1H NMR (CDCl3, 500 MHz):
δ 1.50 – 1.62 (m, 2H), 1.76 – 1.91 (m, 2H), 2.00 – 2.18 (m, 2H), 2.50 – 2.59 (m, 1H), 2.95 – 3.04 (m,
1H), 3.28 – 3.35 (m, 1H), 3.86 – 3.94 (m, 1H), 4.18 (d, J = 14.0 Hz, 1H), 4.37 (d, J = 14.0 Hz, 1H), 8.02
(s, 1H), 8.28 (s, 1H), 8.36 (s, 1H) ppm; 13C NMR (CDCl3, 125 MHz): δ 20.0 (CH2), 26.8 (CH2), 43.5 (CH2),
44.5 (CH2), 50.2 (CH2), 55.7 (CH), 77.8 (CH), 105.2 (C), 121.9 (C), 127.8 (CH), 129.3 (C), 132.6 (CH),
133.3 (C), 147.1 (C), 149.4 (CH), 160.0 (C) ppm.
4.22. (+)-Isohalofuginone 14: In an identical procedure to that described above for (+)-2, (+)-3 (8.0
mg, 0.018 mmol) was heated to reflux in CDCl3 (1 mL) for 24 h affording, after purification by flash
column chromatography (CHCl3/MeOH; 4:1), (+)-14 (5.0 mg, 63%) as an off-white solid. [α]D20 = +34.8
(c = 0.50, CHCl3); with additional data as above for (−)-14.
Supplementary data (proton, carbon NMR spectra and HPLC traces) related to this article can be
found at: http://.
ACKNOWLEDGEMENTS
We thank University College Dublin for a postgraduate scholarship (SS) and Prof. Orna Halevy,
Hebrew University of Jerusalem, Israel and Dr. Kimberly Geoghegan for helpful advice.
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