Design and synthesis of potential mechanism-based inhibitors of the aminotransferase BioA involved in biotin biosynthesis

Article abstract of DOI:10.1021/jo3008435

BioA, a pyridoxal 5′-phosphate (PLP) dependent aminotransferase, catalyzes the second step of biotin biosynthesis, converting 7-keto-8-aminopelargonic acid (KAPA) into 7,8-diaminopelargonic acid (DAPA). Amiclenomycin (ACM) isolated from cultures of different Streptomyces strains is a potent mechanism-based inhibitor of BioA that operates via an aromatization mechanism, irreversibly labeling the PLP cofactor. However, ACM is plagued by inherent chemical stability. Herein we describe the synthesis of four inhibitors, inspired by ACM but containing an allylic amine as the chemical warhead, designed to both improve stability and operate via a complementary Michael addition-pathway upon enzymatic oxidation of the allylic amine substrate to an enimine. Acyclic analogue M-1 contains a terminal olefin as the pro-Michael acceptor. The synthesis of M-1 features an alkyne-zipper reaction and the Overman rearrangement as key synthetic operations. The cyclic analogues M-2/3/4 contain either an endocyclic or exocyclic olefin as the pro-Michael acceptor. These were all prepared using a common strategy employing DIBAL reduction of a precursor bicyclic lactam, followed by in situ Horner-Wadsworth-Emmons (HWE) olefination as the key synthetic steps.

Full text of DOI:10.1021/jo3008435

Article
pubs.acs.org/joc
Design and Synthesis of Potential Mechanism-Based Inhibitors of the
Aminotransferase BioA Involved in Biotin Biosynthesis
Ce Shi and Courtney C. Aldrich*
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: BioA, a pyridoxal 5′-phosphate (PLP) dependent
aminotransferase, catalyzes the second step of biotin biosynthesis,
converting 7-keto-8-aminopelargonic acid (KAPA) into 7,8-
diaminopelargonic acid (DAPA). Amiclenomycin (ACM) isolated
from cultures of different Streptomyces strains is a potent
mechanism-based inhibitor of BioA that operates via an
aromatization mechanism, irreversibly labeling the PLP cofactor.
However, ACM is plagued by inherent chemical stability. Herein
we describe the synthesis of four inhibitors, inspired by ACM but
containing an allylic amine as the chemical warhead, designed to
both improve stability and operate via a complementary Michael
addition-pathway upon enzymatic oxidation of the allylic amine
substrate to an enimine. Acyclic analogue M-1 contains a terminal
olefin as the pro-Michael acceptor. The synthesis of M-1 features
an alkyne-zipper reaction and the Overman rearrangement as key synthetic operations. The cyclic analogues M-2/3/4 contain
either an endocyclic or exocyclic olefin as the pro-Michael acceptor. These were all prepared using a common strategy employing
DIBAL reduction of a precursor bicyclic lactam, followed by in situ Horner−Wadsworth−Emmons (HWE) olefination as the key
synthetic steps.
100 min at pH 8).1 Subsequent transfer of the activated carboxy
group onto acetyl-CoA affords malonyl-CoA, the key
monomeric building block for synthesis of fatty acids. Biotin-
dependent enzymes are also found in other primary and
secondary metabolic pathways including gluconeogenesis,
amino acid catabolism, and polyketide synthesis.1
Bacteria, fungi, and plants synthesize biotin de novo, whereas
mammals must obtain this vital cofactor either from their diet
or from commensal bacteria in the digestive tract. Biotin
auxotrophs of Escherichia coli and Mycobacterium tuberculosis
that can only survive when biotin is supplemented in the
growth medium have been isolated.1,2 The concentration of
biotin in serum from humans is approximately 2 nM, which
could potentially rescue biotin auxotrophs.3 In the case of M.
tuberculosis, Schnappinger and co-workers have shown, using a
novel mycobacterial conditional expression system, that
exogenous host biotin is not sufficient to support bacterial
growth or maintenance in a murine infection model.4
INTRODUCTION
Biotin (vitamin H or B7), a structurally simple bicyclic molecule
comprised of an imidazol-2-one fused to a tetrahydrothiophene
with a pentanoic acid side-chain, is a cofactor required for all
organisms (Figure 1). Biotin is covalently attached via an amide
linkage to the ε-amino group of a conserved lysine residue of
biotin carrier protein domains, which are part of multimeric
enzymes involved in carboxy-transfer reactions.1 In acetyl-CoA
carboxylase, a representative biotin-dependent enzyme, the
biotin cofactor is directly carboxylated at the N-1 position of
■
the imidazol-2-one ring to afford a stable carbamic acid (t1/2
>
The lack of new antibiotics for M. tuberculosis and other
clinically significant Gram negative bacteria coupled with the
dramatic increase of multidrug resistant strains requires new
lead compounds and exploration of other biochemical pathways
outside the conventional antibiotic targets of RNA transcription
and DNA-, protein-, and cell-wall synthesis. On the basis of the
Figure 1. Conversion of KAPA to DAPA catalyzed by BioA. DAPA is
elaborated to biotin by two additional enzymes (BioD and BioB), then
covalently attached to biotin carboxylase carrier protein domains
(BCCP) by an ATP-dependent biotin protein ligase.
Received: April 26, 2012
Published: June 22, 2012
© 2012 American Chemical Society
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confirmed essentiality and inherent bacterial specificity, the
biotin biosynthetic pathway represents an attractive target for
the development of new antibacterial agents.
extensive previous studies by Silverman and others on related
mechanism-based inhibitors of PLP-dependent enzymes.10 As
illustrated in Figure 2B using M-4 as a representative example,
the inhibitor is expected to behave like a normal substrate
undergoing transamination with the enzyme bound PLP
cofactor to afford the inhibitor−PLP aldimine I. Subsequent
redox isomerization (i.e., a 1,3-prototropic shift) mediated by a
general base (Lys283 in BioA from M. tuberculosis) provides the
electrophilic enimine III via quinonoid intermediate II.
Enimine III is a Michael acceptor and can react with the
general base to afford adduct IV.
BioA, a pyridoxal 5′-phosphate (PLP) dependent amino-
transferase, catalyzes the second step of biotin biosynthesis,
converting 7-keto-8-aminopelargonic acid (KAPA) into 7,8-
diaminopelargonic acid (DAPA) using S-adenosylmethionine
(SAM) as the amino donor (Figure 1).5 Amiclenomycin
(ACM), isolated from cultures of different Streptomyces strains,6
and its simplified amino-alcohol analog7 (ACM−OH, Figure
2A) are potent inhibitors of BioA.8 Structurally, both ACM and
ACM−OH bear a symmetrical cis-1,4-cyclohexadiene ring that
acts as the chemical warhead, resulting in irreversible
inactivation of the PLP cofactor of BioA via an aromatization
mechanism.5b,9 Interestingly, ACM and ACM−OH exhibit
selective activity against M. tuberculosis from over 40 bacterial
strains evaluated.8
While mechanistically and structurally intriguing, amicleno-
mycin suffers from inherent poor chemical stability, which
results in rapid aromatization to an inactive aniline derivative.8
Consequently, we sought to develop inhibitors wherein the cis-
1,4-cyclohexadiene warhead was replaced with a series of acyclic
and cyclic warheads containing chemically stable allylic amines
capable of inactivating BioA via a Michael addition mechanism
(see Figure 2A, M-1/2/3/4). This inhibitor design is based on
RESULTS AND DISCUSSION
■
Acyclic analogue M-1 was designed to impart flexibility in the
inhibitor scaffold to allow optimal positioning of the β-carbon
of the Michael acceptor with the incipient nucleophile.
Retrosynthetically, we envisioned M-1 could be derived from
a trans-allylic imidate by the Overman rearrangement transform
(Scheme 1).11 The palladium-catalyzed version using either
Scheme 1. Synthesis of M-1
(R)- or (S)-COP-Cl would allow access to either enantio-
mer.11c Further disconnection affords a terminal alkyne that in
turn can be prepared by isomerization of commercially available
3-alkyn-1-ol.
The synthesis of acyclic analogue M-1 began with the
Brown−Yamashita alkyne isomerization of 3-octyn-1-ol 1 into
the more thermodynamically stable terminal alkyne (as a result
of the greater Csp−H versus Csp−Csp3 bond strength)
employing ethylenediamine and NaH followed by TBS
protection to afford 2 (Scheme 1).12 Deprotonation of the
alkyne and addition to formaldehyde provided propargyl
alcohol 3. Stereospecific reduction of 3 by Red-Al furnished
trans-allylic alcohol 4, which was converted to the correspond-
ing key trichloroacetimidate intermediate 5 by treatment with
trichloroacetonitrile and DBU. 1,3-Transposition was accom-
plished by refluxing 5 in xylene to furnish allylic amine 6.11
Finally, sequential removal of TBS and trichloroacetyl groups
provided M-1 in good overall yield (Scheme 1).
Figure 2. (A) Design of novel Michael addition-based inhibitors; (B)
proposed Michael addition-based mechanism of inhibition.
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Since cyclic analogues M-2/3/4 share common structural
features, a general homologation strategy was developed.
Retrosynthetically, we envisaged that all analogs could be
derived from the corresponding bridged lactams (Figure 3).13
the subsequent decarboxylation went smoothly under very mild
condition with quantitative yield, the coupling reaction of
Meldrum’s acid with the amino acid intermediate derived from
Vince lactam 7b suffered from epimerization as a result of the
highly activated acyl-pyridinium intermediate. Lastly, a
Horner−Wadsworth−Emmons (HWE) olefination−conjugate
reduction strategy was evaluated for the homologation. DIBAL-
H reduction of 7b afforded an intermediate hemiaminal in
equilibrium with the open-chained amino-aldehyde, which was
reacted with triethyl phosphonoacetate to afford the conjugate
ester 10 with exclusive trans configuration in 81% isolated yield
over two steps. CuBr-catalyzed conjugate reduction furnished
the desired ester intermediate 11 in quantitative yield. As
observed previously,16 the use of cyclohexene in large excess
was essential to prevent reduction of the isolated alkene.
Finally, reduction of ester 11, followed by Boc deprotection
gave M-2 as its hydrochloride salt. Overall, the HWE
olefination−conjugate reduction strategy required the shortest
synthetic route and provided the highest isolated product yield
among the three routes evaluated and was subsequently used
for the synthesis of both M-3 and M-4.
Figure 3. Retrosynthetic analysis of M-2/3/4.
The synthesis of M-3 bearing an exocyclic methylene also
started from Vince lactam 7a (Table 1). Following a previous
The advantages of utilizing the lactams as starting points
include: (1) the stereochemistry at the bridge points ensures
the cis configuration once the lactam is opened; (2) there are
multiple routes to convert lactams into corresponding amino
alcohol derivatives, which allows us to find the optimal
conditions for side chain homologation; (3) these lactams are
either commercially available or can be synthesized conven-
iently from reported procedures.13
Table 1. Optimization of Olefination
Analogue M-2 can be synthesized from commercially
available Vince lactam 7a, which was converted to the N-Boc
derivative 7b as reported in 95% yield.14 The Boc group
enhances the electrophilicity of the amide enabling ring-
opening under substantially milder conditions than 7a. Three
homologation strategies were tested using 7b as the starting
material (Scheme 2). We first followed the reported procedures
to synthesize the substituted malonate ester 8 from 7b in 4
steps.13a LiCl-mediated decarboxylation of 8 did lead to the
desired monoester 11, however, the reaction required high
temperature (∼160 °C), at which loss of the Boc group became
a major competitive side reaction. To find a better
decarboxylation method, DCC-promoted coupling with Mel-
drum’s acid followed by reduction was performed.15 Although
report, the 6-keto Vince lactam 12 was synthesized
conveniently in 5 steps.13b,c Four sets of reaction conditions
were then screened for the olefination step (Table 1). The
conventional Wittig (entry 1) and Peterson (entry 2)
Scheme 2. Synthesis of M-2
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conditions led to incomplete reaction and poor isolated yields.
On the other hand, the Tebbe-type olefination conditions
(entries 3 and 4) provided a fairly good yield of 13.
Replacement of Zn and CH2Br2 with Nysted’s reagent (entry
4) both substantially shortened the reaction time and improved
the isolated yield to 88%. Therefore, the TiCl4 mediated
olefination using Nysted’s reagent (entry 4) became the
standard condition for synthesis of exocyclic methylene analogs
M-3/4.
lactam 18 was first synthesized following the reported
procedures from 2-pyridone 17.13d Methylenation employing
Nysted’s reagent afforded 19. Next, the benzyl protecting group
was exchanged for a Boc group (19→20). Elaboration of 20 to
22 was accomplished in four steps by sequential DIBAL-H
reduction, HWE olefination, conjugate reduction, and LAH
ester reduction in 67% overall yield over 4 steps (90.5% average
yield per step) with a single purification. Treatment of 22 with
4 N HCl in dioxane furnished M-4, which was isolated as the
hydrochloride salt in 89% by recrystallization from MeOH−
Et2O.
Compounds M-1/2/3/4 were evaluated for enzyme
inhibition using a continuous coupled assay under initial
velocity conditions.17 Unfortunately, only analogue M-2
showed any enzyme inhibition of BioA providing a modest
IC50 value of 57 μM (Table 1). However, M-2 did not exhibit
time-dependent inhibition as would be expected for an
irreversible inhibitor. These results show that BioA is less
promiscuous than originally anticipated. Our recently disclosed
crystal structure of BioA with a dihydropyridone inhibitor,
which acts as an aromatization mechanism-based inactivator,8
suggests that inhibitors should be relatively flat. Indeed, the
cyclohexadiene warhead of amiclenomycin (ACM) is planar.
With an efficient and reliable method for the synthesis of 13
established, we now sought to complete the synthesis of M-3
(Scheme 3). First, protecting group exchange was performed
Scheme 3. Synthesis of M-3
Table 2. Inhibition Activity of M-1−4 against BioA
(13→14). Chemoselective reductive removal of the N-benzyl
protecting group, without disturbing the sensitive exocyclic
olefin, was successfully accomplished employing Birch con-
ditions. The resultant intermediate amide was then Boc-
protected to afford 14. It should be noted that the relatively
harsh reaction conditions required in the preparation of bicyclic
lactam 12 necessitate a benzyl protecting group. Bicyclic amide
14 was elaborated to M-3 in analogy to the synthesis of M-2 by
sequential DIBAL-H reduction, HWE olefination, conjugate
reduction, LAH ester reduction, and deprotection of the Boc
group.
The synthesis of M-4 bearing a 6-membered ring and
exocyclic methylene proceeded as shown in Scheme 4. 6-Keto
In conclusion, we have synthesized and biochemically
characterized a series of compounds designed to inhibit BioA.
Overall, the synthesis of M-1 is flexible and redox economical18
using isomerizations to manipulate the functionality. A
common strategy was developed for the synthesis of analogues
M-2/3/4 featuring DIBAL-H reduction of a bicyclic lactam to
an intermediate hemiaminal and subsequent in situ Horner−
Wadsworth−Emmons olefination. Introduction of the exocyclic
methylene in analogues M-3/4 was optimally performed using
the Nysted reagent. Biochemical evaluation of all four inhibitors
indicate that planarity of the inhibitor is likely important since
only cyclopentene M-2 demonstrated activity.
Scheme 4. Synthesis of M-4
EXPERIMENTAL SECTION
■
General Methods. All chemical reactions were performed under
an inert atmosphere of dry Ar or N2 in oven-dried (150 °C) glassware.
1H and 13C NMR spectra were recorded on a 600 MHz spectrometer.
Proton chemical shifts are reported in ppm from an internal standard
of residual chloroform (7.26 ppm) or methanol (3.31 ppm), and
carbon chemical shifts are reported using an internal standard of
residual chloroform (77.0 ppm) or methanol (49.1 ppm). Proton
chemical data are reported as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet,
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br = broad), coupling constant, integration. Melting points were
measured on a melting point apparatus and uncorrected. High
resolution mass spectra were obtained on a TOF II TOF/MS
instrument equipped with either an ESI or APCI interface. TLC
analyses were performed on TLC silica gel plates and were visualized
with UV light, iodine chamber, 10% sulfuric acid or 10% PMA
solution. Purifications were performed by flash chromatography on
silica gel (60A). Enzyme inhibition assays were performed on an M5e
multimode plate reader.
Materials. An anhydrous solvent dispensing system using 2 packed
columns of neutral alumina was used for drying THF, Et2O, and
CH2Cl2 while 2 packed columns of molecular sieves were used to dry
DMF and the solvents were dispensed under argon. 7b,14 8,13a 12,13b,c
and 1813d were prepared as described.
HRMS (APCI+) calcd for C15H31O2Si [M + H]+ 271.2088, found
271.2083 (error 1.8 ppm).
(E)-9-(tert-Butyldimethylsilyloxy)non-2-en-1-ol (4). To a sol-
ution of 3 (1.95 g, 7.2 mmol, 1 equiv) in Et2O (40 mL) at 0 °C, was
added Red-Al (65 wt % in toluene, 6.5 mL, 19.5 mmol, 3 equiv)
dropwise over 15 min. The solution was stirred at 0 °C for 6 h and
quenched by the dropwise addition of MeOH (0.5 mL) followed by 1
N aqueous potassium sodium tartrate (25 mL). The mixture was
gradually warmed to 25 °C and stirred for 0.5 h. The aqueous layer
was then extracted with Et2O (3 × 20 mL) and the combined organic
layers were dried (MgSO4) and concentrated. Purification by silica gel
column chromatography (5:95 EtOAc−hexanes to 10:90 EtOAc−
hexanes) afforded the title compound (1.83 g, 93%) as a colorless oil:
1
Rf = 0.23 (20:80 EtOAc−hexanes); H NMR (CDCl3, 600 MHz) δ
tert-Butyldimethyl(oct-7-ynyloxy)silane (2). To ethylene-1,2-
diamine (30 mL) at 0 °C, was added NaH (2.54 g, 60 wt % in mineral
oil, 64 mmol, 4 equiv) in one portion. The mixture was stirred at 0 °C
for 5 min, 1 h at 25 °C, then heated at 65 °C for 1 h. The mixture was
cooled to 45 °C and 3-octyn-1-ol (2.0 g, 16 mmol, 1 equiv) was added
dropwise over 15 min. After the addition, the reaction was heated at 65
°C and stirred for 1 h. The mixture was cooled down to 0 °C and
quenched by the successive cautious addition of H2O (25 mL) and 1
N aqueous HCl (25 mL). The quenched reaction mixture was diluted
with 1 N aqueous HCl (25 mL) and extracted with Et2O (3 × 25 mL).
The combined organic layers were washed with 1 N aqueous HCl (20
mL), saturated aqueous NaCl (20 mL), dried (MgSO4) and
concentrated. Purification by silica gel column chromatography
(10:90 EtOAc−hexanes to 20:80 EtOAc−hexanes) afforded 7-octyn-
1-ol (1.5 g, 72%) as a colorless oil: Rf = 0.15 (1:4 EtOAc−hexanes);
1H NMR (CDCl3, 600 MHz) δ 1.28−1.31 (m, 2H), 1.32−1.38 (m,
2H), 1.40−1.53 (m, 4H), 1.90 (t, J = 2.4 Hz, 1H), 2.12 (td, J = 7.2, 2.4
Hz, 2H), 2.34 (br s, 1H, OH), 3.55 (t, J = 6.6 Hz, 2H); 13C NMR
(CDCl3, 150 MHz) δ 18.5, 25.4, 28.6, 28.7, 32.7, 62.8, 68.4, 84.8. All
data are consistent with reported literature values.12b
To a solution of 7-octyn-1-ol (1.0 g, 7.9 mmol, 1 equiv) prepared
above, DMAP (98 mg, 0.79 mmol, 0.1 equiv) and imidazole (806 mg,
11.8 mmol, 1.5 equiv) in THF (10 mL) at 0 °C, was added a stock
solution of tert-butyldimethylsilyl chloride in THF (4.4 M, 2.0 mL, 8.8
mmol, 1.1 equiv) over 5 min. The mixture was stirred for 1 h at 0 °C
and quenched by addition of saturated aqueous NH4Cl (15 mL). The
aqueous layer was extracted with Et2O (3 × 15 mL). The combined
organic layers were washed with H2O (20 mL), saturated aqueous
NaCl (20 mL), dried (MgSO4) and concentrated. Purification by silica
gel column chromatography (5:95 EtOAc−hexanes to 10:90 EtOAc−
hexanes) afforded the title compound (1.8 g, 95%) as a colorless oil: Rf
= 0.65 (10:90 EtOAc−hexanes); 1H NMR (CDCl3, 600 MHz) δ 0.04
(s, 6H), 0.88 (s, 9H), 1.33 (p, J = 7.2 Hz, 2H), 1.40 (p, J = 7.2 Hz,
2H), 1.50−1.54 (m, 4H), 1.93 (t, J = 2.4 Hz, 1H), 2.17 (td, J = 7.2, 2.4
Hz, 2H), 3.60 (t, J = 6.6 Hz, 2H); 13C NMR (CDCl3, 150 MHz) δ
−5.3, 18.42, 18.44, 25.3, 26.0, 28.5, 28.6, 32.7, 63.1, 68.1, 84.7; HRMS
(APCI+) calcd for C14H29OSi [M + H]+ 241.1982, found 241.1985
(error 1.2 ppm).
0.03 (s, 6H), 0.88 (s, 9H), 1.29−1.30 (m, 4H), 1.36−1.38 (m, 2H),
1.48−1.50 (m, 2H), 1.67 (br s, 1H, OH), 2.02 (q, J = 6.6 Hz, 2H),
3.58 (t, J = 6.6 Hz, 2H), 4.05 (d, J = 6.0 Hz, 2H), 5.58−5.75 (m, 2H);
13C NMR (CDCl3, 150 MHz) δ −5.4, 18.2, 25.5, 25.9, 28.8, 29.0, 32.0,
32.7, 63.2, 63.6, 128.8, 133.2; HRMS (APCI+) calcd for C15H33O2Si
[M + H]+ 273.2244, found 273.2251 (error 2.6 ppm).
(E)-9-(tert-Butyldimethylsilyloxy)non-2-enyl 2,2,2-trichloroa-
cetimidate (5). To a solution of 4 (272 mg, 1 mmol, 1 equiv) in
CH2Cl2 (10 mL) at 0 °C, were sequentially added DBU (174 μL, 1.15
mmol, 1.15 equiv) and CCl3CN (147 μL, 1.46 mmol, 1.46 equiv)
dropwise. The solution was stirred at 0 °C for 2 h. TLC indicated
complete conversion and the reaction was concentrated in vacuo.
Purification by silica gel column chromatography (hexanes to 10:90
EtOAc−hexanes) afforded the title compound (395 mg, 95%) as a
1
colorless oil: Rf = 0.45 (10:90 EtOAc−hexanes); H NMR (CDCl3,
600 MHz) δ 0.04 (s, 6H), 0.88 (s, 9H), 1.30−1.32 (m, 4H), 1.39−1.41
(m, 2H), 1.49−1.51 (m, 2H), 2.07 (q, J = 7.2 Hz, 2H), 3.59 (t, J = 6.6
Hz, 2H), 4.73 (d, J = 6.6 Hz, 2H), 5.67 (dt, J = 15.0, 6.6 Hz, 1H), 5.86
(dt, J = 15.0, 6.6 Hz, 1H), 8.26 (br s, 1H); 13C NMR (CDCl3, 150
MHz) δ −5.0, 18.6, 25.9, 26.2, 29.0, 29.1, 32.5, 33.0, 63.4, 70.2, 91.8,
123.2, 137.3, 162.8; HRMS (APCI+) calcd for C17H33Cl3NO2Si [M +
H]+ 416.1341, found 416.1351 (error 2.4 ppm).
( )-N-[9-(tert-Butyldimethylsilyloxy)non-1-en-3-yl]-2,2,2-tri-
chloroacetamide (6). A solution of 5 (300 mg, 0.74 mmol, 1 equiv)
in o-xylene (2 mL) was heated at 130 °C for 6 h. TLC indicated
complete conversion and the reaction was concentrated in vacuo.
Purification by silica gel column chromatography (hexanes to 10:90
EtOAc−hexanes) afforded the title compound (265 mg, 88%) as a
1
colorless oil: Rf = 0.40 (10:90 EtOAc−hexanes); H NMR (CDCl3,
600 MHz) δ 0.04 (s, 6H), 0.88 (s, 9H), 1.27−1.40 (m, 7H), 1.48−1.51
(m, 1H), 1.59−1.61 (m, 1H), 1.63−1.68 (m, 1H), 3.59 (t, J = 6.6 Hz,
2H), 4.40 (p, J = 6.6 Hz, 1H), 5.18 (d, J = 10.8 Hz, 1H), 5.23 (d, J =
16.8 Hz, 1H), 5.79 (ddd, J = 16.8, 10.8, 5.4 Hz, 1H), 6.50 (br d, J = 7.2
Hz, 1H, NH); 13C NMR (CDCl3, 150 MHz) δ −5.0, 18.6, 25.7, 25.8,
26.2, 29.3, 32.9, 34.6, 53.8, 63.3, 93.1, 116.3, 136.9, 161.4; HRMS
(ESI−) calcd for C17H31Cl3NO2Si [M − H]− 414.1195, found
414.1198 (error 0.7 ppm).
( )-7-Aminonon-8-en-1-ol Hydrochloride Salt (M-1). To a
solution of 6 (215 mg, 0.52 mmol, 1 equiv) in THF (3 mL) at 0 °C,
was added HF·Py (1 mL) dropwise. The solution was stirred at 0 °C
for 2 h when TLC indicated complete conversion. The reaction was
diluted with EtOAc (20 mL) and washed with saturated aqueous
NH4Cl (10 mL). The aqueous layer was extracted with EtOAc (3 × 10
mL). The combined organic layers were dried (MgSO4) and
concentrated. Purification by silica gel column chromatography
(5:95 EtOAc−hexanes to 50:50 EtOAc−hexanes) afforded
( )-2,2,2-trichloro-N-(9-hydroxynon-1-en-3-yl)acetamide (134 mg,
95%) as a colorless oil: Rf = 0.40 (50:50 EtOAc−hexanes); 1H
NMR (CD3OD, 600 MHz) δ 1.36 (br s, 6H), 1.52 (t, J = 6.0 Hz, 2H),
1.63−1.67 (m, 2H), 3.53 (t, J = 6.6 Hz, 2H), 4.33 (p, J = 7.2 Hz, 1H),
4.84 (br s, 1H), 5.11 (d, J = 10.8 Hz, 1H), 5.19 (d, J = 16.8 Hz, 1H),
5.85 (ddd, J = 16.8, 10.8, 5.4 Hz, 1H), 8.68 (d, J = 7.2 Hz, 1H); 13C
NMR (CD3OD, 150 MHz) δ 26.9, 27.3, 30.2, 33.6, 35.1, 55.7, 63.0,
94.4, 116.0, 138.9, 163.7; HRMS (APCI+) calcd for C11H19Cl3NO2
[M + H]+ 302.0476, found 302.0485 (error 3.0 ppm).
9-(tert-Butyldimethylsilyloxy)non-2-yn-1-ol (3). To a solution
of 2 (1.2 g, 5.0 mmol, 1 equiv) in THF (20 mL) at −78 °C, was added
n-BuLi (2.5 M in hexanes, 2.4 mL., 6.0 mmol, 1.2 equiv) dropwise over
15 min. The mixture was stirred at −78 °C for 30 min and
paraformaldehyde (980 mg, 32.5 mmol, 6.5 equiv) was added in one
portion. The reaction was gradually warmed to 25 °C over 3 h. TLC
indicated complete conversion of starting material and the reaction
was then quenched by addition of saturated aqueous NH4Cl (25 mL).
The aqueous layer was extracted with Et2O (3 × 20 mL). The
combined organic layers were washed with H2O (20 mL), saturated
aqueous NaCl (20 mL), dried (MgSO4) and concentrated. Purification
by silica gel column chromatography (5:95 EtOAc−hexanes to 10:90
EtOAc−hexanes) afforded the title compound (1.3 g, 85%) as a
1
colorless oil: Rf = 0.25 (20:80 EtOAc−hexanes); H NMR (CDCl3,
600 MHz) δ 0.03 (s, 6H), 0.88 (s, 9H), 1.30−1.40 (m, 4H), 1.47−1.53
(m, 4H), 1.86 (br s, 1H, OH), 2.19 (tt, J = 7.2, 1.8 Hz, 2H), 3.59 (t, J
= 6.6 Hz, 2H), 4.22 (t, J = 1.8 Hz, 2H); 13C NMR (CDCl3, 150 MHz)
δ −5.1, 18.6, 18.9, 25.5, 26.2, 28.8, 28.9, 32.9, 51.4, 63.4, 78.7, 86.5;
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To a solution of ( )-2,2,2-trichloro-N-(9-hydroxynon-1-en-3-yl)-
acetamide prepared above (134 mg, 0.50 mmol, 1 equiv) in toluene (2
mL) at −78 °C, was added a 1 M DIBAL-H solution in CH2Cl2 (2
mL, 4 equiv) dropwise over 10 min. The solution was stirred at −78
°C for 1 h, then the reaction was quenched by the dropwise addition
of MeOH (0.5 mL) and 1 N aqueous potassium sodium tartrate (25
mL). The mixture was gradually warmed to 25 °C and stirred for 30
min. The aqueous layer was extracted with EtOAc (3 × 20 mL) and
the combined organic layers were dried (MgSO4) and concentrated.
The crude material was acidified with 4 N HCl in dioxane (2 mL) and
concentrated again. Recrystallization from MeOH−Et2O (1:20, 5 mL)
afforded the title compound (75 mg, 78%) as an off white solid: mp =
2.31 (t, J = 7.8 Hz, 2H), 2.31−2.60 (m, 2H), 4.11 (q, J = 7.2 Hz, 2H),
4.54 (br s, 1H), 4.66 (br s, 1H), 5.65 (d, J = 5.4 Hz, 1H), 5.77 (d, J =
5.4 Hz, 1H); 13C NMR (CDCl3, 150 MHz) δ 14.3, 28.6, 31.6, 32.7,
38.6, 44.0, 56.6, 60.6, 79.4, 132.3, 137.1, 155.4, 173.7; HRMS (APCI+)
calcd C15H26NO4 [M + H]+ 284.1856, found 284.1857 (error 0.4
ppm).
(
)-cis-3-(−4-Aminocyclopent-2-enyl)propan-1-ol Hydro-
chloride Salt (M-2). To a solution of 11 (140 mg, 0.5 mmol, 1
equiv) in THF (5 mL) at 0 °C, was added LiAlH4 (28.4 mg, 0.75
mmol, 1.5 equiv) in one portion. The reaction was stirred at 0 °C for
0.5 h and quenched by the successive dropwise addition of MeOH
(0.5 mL) and saturated aqueous potassium sodium tartrate (20 mL).
The mixture was warmed up to 25 °C and stirred for 0.5 h. The
aqueous layer was extracted with EtOAc (3 × 20 mL). The combined
organic layers were washed with H2O (20 mL), saturated NaCl
solution (20 mL), dried (MgSO4) and concentrated. Purification by
silica gel column chromatography (10:90 EtOAc−hexanes to 50:50
EtOAc−hexanes) afforded ( )-tert-butyl cis-4-(3-hydroxypropyl)-
cyclopent-2-enylcarbamate (100 mg, 83%) as a colorless foam: Rf =
1
89−91 °C; H NMR (CD3OD, 600 MHz) δ 1.35−1.40 (m, 6H),
1.51−1.54 (m, 2H), 1.63−1.66 (m, 1H), 1.73−1.75 (m, 1H); 3.54 (t, J
= 6.6 Hz, 2H), 3.68 (q, J = 5.4 Hz, 1H), 5.41 (d, J = 10.8 Hz, 1H), 5.43
(d, J = 17.4 Hz, 1H), 5.82 (ddd, J = 17.4, 10.8, 5.4 Hz, 1H); 13C NMR
(CD3OD, 150 MHz) δ 26.4, 26.8, 30.1, 33.5, 34.1, 55.5, 63.0, 121.3,
135.4; HRMS (ESI+) calcd for C9H20NO [M + H]+ 158.1539, found
158.1544 (error 3.2 ppm).
1
0.35 (50:50 EtOAc−hexanes); H NMR (CDCl3, 600 MHz) δ 1.06−
(
)-Ethyl cis-(E)-3-(4-{[(tert-butoxy)carbonyl]amino}-
cyclopent-2-enyl)acrylate (10). To a solution of 7b (300 mg, 1.4
mmol, 1 equiv) in toluene (4 mL) at −78 °C, was added dropwise 1 M
DIBAL-H in toluene (2.2 mL, 2.2 mmol, 1.5 equiv) over 15 min. The
solution was stirred at −78 °C for 1 h and quenched by the addition of
MeOH (0.5 mL) and saturated aqueous potassium sodium tartrate (20
mL). The mixture was gradually warmed to 25 °C and stirred for 0.5 h.
The aqueous layer was extracted with EtOAc (3 × 20 mL). The
combined organic layers were washed with H2O (20 mL), saturated
aqueous NaCl (20 mL), dried (MgSO4) and concentrated to afford
( )-tert-butyl 3-hydroxy-2-azabicyclo[2.2.1]hept-5-ene-2-carboxylate
as a mixture of diastereomers epimeric at C-3, which was used
directly in the next step without further purification.
1.08 (m, 1H), 1.31−1.35 (m, 1H), 1.43 (s, 9H), 1.47−1.61 (m, 3H),
2.57−2.60 (m, 2H), 3.63 (t, J = 6.6 Hz, 2H), 4.53 (br s, 1H), 4.66 (br
s, 1H), 5.63 (d, J = 4.8 Hz, 1H), 5.80 (d, J = 4.8 Hz, 1H); 13C NMR
(CDCl3, 150 MHz) δ 28.7, 31.1, 32.9, 39.1, 44.4, 56.7, 63.1, 79.5,
131.5, 137.9, 155.5; HRMS (APCI+) calcd for C13H24NO3 [M + H]+
242.1751, found 242.1761 (error 4.1 ppm).
( )-tert-Butyl cis-4-(3-hydroxypropyl)cyclopent-2-enylcarbamate
prepared above (48 mg, 0.2 mmol, 1 equiv) was dissolved in 4 N
HCl in dioxane (2 mL, 8.0 mmol, 40 equiv) at 0 °C. The solution was
stirred at 0 °C for 1 h, then concentration in vacuo. Recrystallization
from MeOH−Et2O (1:20, 5 mL) afforded the title compound (31 mg,
1
89%) as an off-white solid: mp = 123−125 °C; H NMR (CD3OD,
To a solution of triethylphosphonoacetate (0.28 mL, 1.68 mmol, 1.2
equiv) in toluene (4 mL) at 0 °C, was added NaH (60 wt % in mineral
oil, 72 mg, 1.68 mmol, 1.2 equiv) in one portion. The mixture was
stirred at 0 °C for 30 min, then a solution of ( )-tert-butyl 3-hydroxy-
2-azabicyclo[2.2.1]hept-5-ene-2-carboxylate prepared above in toluene
(2 mL) was added dropwise. The reaction was gradually warmed to 25
°C and stirred for 15 h. TLC indicated complete conversion and the
reaction was partitioned between saturated aqueous NH4Cl (20 mL)
and EtOAc (20 mL). The aqueous layer was extracted with EtOAc (3
× 20 mL). The combined organic layers were washed with H2O (20
mL), saturated aqueous NaCl (20 mL), dried (MgSO4) and
concentrated. Purification by silica gel column chromatography
(hexanes to 10:90 EtOAc−hexanes) afforded the title compound
(334 mg, 81%) as a colorless foam: Rf = 0.55 (20:80 EtOAc−hexanes);
1H NMR (CDCl3, 600 MHz) δ 1.25 (t, J = 6.6 Hz, 3H), 1.35 (dt, J =
600 MHz) δ 1.35 (p, J = 6.6 Hz, 1H), 1.43−1.46 (m, 1H), 1.47−1.64
(m, 3H), 2.62−2.67 (m, 1H), 2.76−2.78 (m, 1H), 3.57 (t, J = 6.0 Hz,
2H), 4.25−4.26 (m, 1H), 5.76 (d, J = 5.4 Hz, 1H), 6.11 (d, J = 5.4 Hz,
1H); 13C NMR (CD3OD, 150 MHz) δ 31.9, 33.3, 36.6, 46.5, 58.1,
62.9, 127.9, 143.2; HRMS (ESI+) calcd for C8H16NO [M + H]+
142.1226, found 142.1220 (error 4.2 ppm).
( )-2-Benzyl-6-methylene-2-azabicyclo[2.2.1]heptan-3-one
(13). To a suspension of Nysted reagent (20 wt %, 8.4 g, 3.7 mmol, 3.0
equiv) at 0 °C, was added a 1 M solution of TiCl4 in CH2Cl2 (3.6 mL,
3.6 mmol, 3.0 equiv) dropwise over 10 min. The mixture was stirred at
0 °C for additional 1 h then a solution of 12 (264 mg, 1.2 mmol, 1.0
equiv) in THF (4 mL) was added dropwise over 10 min. The reaction
was gradually warmed to 25 °C and stirred for 3 h at 25 °C when TLC
indicated complete conversion. The reaction was cooled to 0 °C and
quenched by the careful dropwise addition of 1 N aqueous HCl (20
mL). The aqueous layer was extracted with EtOAc (3 × 20 mL) and
the combined organic layers were washed with H2O (20 mL),
saturated aqueous NH4Cl (20 mL), dried (MgSO4) and concentrated.
Purification by silica gel column chromatography (10:90 EtOAc−
hexanes to 50:50 EtOAc−hexanes) afforded the title compound (230
12.0, 6.0 Hz, 1H), 1.40 (s, 9H), 2.61−2.65 (m, 1H), 3.35−3.37 (m,
1H), 4.16 (q, J = 6.6 Hz, 2H), 4.59 (br s, 1H), 4.72 (br s, 1H), 5.72−
5.78 (m, 3H), 6.86 (dd, J = 15.6, 7.2 Hz, 1H); 13C NMR (CDCl3, 150
MHz) δ 14.5, 28.6, 38.5, 46.5, 56.7, 60.6, 79.8, 120.8, 134.0, 134.5,
151.0, 155.3, 166.8; HRMS (APCI+) calcd for C15H24NO4 [M + H]+
282.1700, found 282.1708 (error 2.8 ppm).
1
mg, 88%) as a colorless foam: Rf = 0.45 (50:50 EtOAc−hexanes); H
NMR (CDCl3, 600 MHz) δ 1.54 (d, J = 9.6 Hz, 1H), 1.96−1.99 (m,
1H), 2.21 (dt, J = 16.2, 2.4 Hz, 1H), 2.40 (dt, J = 16.2, 2.4 Hz, 1H),
2.89 (d, J = 2.4 Hz, 1H), 3.70 (d, J = 15.0 Hz, 1H), 3.72−3.73 (m,
1H), 4.75 (d, J = 15.0 Hz, 1H), 4.93 (br s, 1H), 5.03 (t, J = 2.4 Hz,
1H), 7.22−7.27 (m, 3H), 7.30−7.33 (m, 2H); 13C NMR (CDCl3, 150
MHz) δ 31.8, 40.4, 44.3, 45.7, 64.4, 107.5, 127.7, 128.3, 128.8, 137.4,
146.1, 178.6; All data are consistent with reported literature values.13c
( )-Ethyl cis-3-(4-{[(tert-butoxy)carbonyl]amino}cyclopent-
2-enyl)propanoate (11). To a solution of 10 (281 mg, 1.0 mmol,
1.0 equiv), CuCl (74 mg, 0.75 mmol, 0.75 equiv) and cyclohexene (0.4
mL, 4.0 mmol, 4.0 equiv) in MeOH (20 mL) at −78 °C, was added
NaBH4 (189 mg, 5.0 mmol, 5.0 equiv). The reaction was stirred at
−78 °C for 1 h, during which time the solution went from brown to
green. MS (APCI+) indicated complete conversion. The reaction was
concentrated in vacuo while the flask was still cold. The crude product
was then partitioned between saturated aqueous NH4Cl (30 mL) and
EtOAc (30 mL). The aqueous layer was extracted with EtOAc (3 × 20
mL). The combined organic layers were washed with saturated
aqueous NaCl, dried (MgSO4) and concentrated. Purification by silica
gel column chromatography (5:95 EtOAc−hexanes to 20:80 EtOAc−
hexanes) afforded the title compound (280 mg, 99%) as a colorless
foam: Rf = 0.55 (20:80 EtOAc−hexanes); 1H NMR (CDCl3, 600
MHz) δ 1.07 (dt, J = 12.0, 6.0 Hz, 1H), 1.24 (t, J = 7.2 Hz, 3H), 1.43
(s, 9H), 1.64 (dt, J = 15.6, 7.8 Hz, 1H), 1.76 (dt, J = 15.6, 7.8 Hz, 1H),
(
)-tert-Butyl 6-methylene-3-oxo-2-azabicyclo[2.2.1]-
heptane-2-carboxylate (14). To a solution of t-BuOH (1.0 mL,
10.6 mmol, 5.6 equiv) in THF (10 mL) at −78 °C, was condensed
NH3 (approximately 10 mL). Na0 (200 mg, 8.7 mmol, 4.6 equiv) was
added to the solution in portions to afford a dark blue solution. Next, a
solution of 13 (400 mg, 1.88 mmol, 1 equiv) in THF (10 mL) was
added dropwise over 20 min. After addition, the reaction was stirred at
−78 °C for 15 min and −30 °C for 30 min. The reaction was then
cooled to −78 °C and quenched by the dropwise addition of AcOH (1
mL). The mixture was gradually warmed to 25 °C. The reaction was
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filtered over Celite and concentrated to afford crude 6-methylene-2-
azabicyclo[2.2.1]heptan-3-one, which was used directly in the next
step without further purification.
EtOAc−hexanes); 1H NMR (CDCl3, 600 MHz) δ 1.65−1.74 (m,
3H), 1.86−1.91 (m, 1H), 2.41 (dq, J = 16.8, 2.4 Hz, 1H), 2.57 (dq, J =
16.8, 2.4 Hz, 1H), 2.75 (t, J = 3.0 Hz, 1H), 3.74 (t, J = 3.0 Hz, 1H),
4.37 (d, J = 15.0 Hz, 1H), 4.70−4.76 (m, 3H), 7.24−7.26 (m, 3H),
7.28−7.31 (m, 2H); 13C NMR (CDCl3, 150 MHz) δ 23.6, 28.0, 32.3,
39.4, 47.4, 59.9, 107.1, 127.3, 128.0, 128.3, 137.2, 144.8, 174.8; HRMS
(APCI+) calcd for C15H18NO [M + H]+ 228.1383, found 228.1375
(error 3.5 ppm).
To a solution of 6-methylene-2-azabicyclo[2.2.1]heptan-3-one
obtained from the previous step and DMAP (114 mg, 0.94 mmol,
0.5 equiv) in CH3CN (10 mL) at 25 °C, was added (Boc)2O (615 mg,
2.8 mmol, 1.5 equiv) in one portion. The mixture was stirred at 25 °C
for 2 h, then concentrated in vacuo. Purification by silica gel column
chromatography (5:95 EtOAc−hexanes to 20:80 EtOAc−hexanes)
afforded the title compound (326 mg, 78%) as a colorless foam: Rf =
0.35 (20:80 EtOAc−hexanes); 1H NMR (CDCl3, 600 MHz) δ 1.49 (s,
9H), 1.58 (dt, J = 10.6, 1.2 Hz, 1H), 2.04−2.07 (m, 1H), 2.35−2.38
(m, 1H), 2.46−2.49 (m, 1H), 2.91 (br d, J = 2.4 Hz, 1H), 4.65 (d, J =
1.8 Hz, 1H), 4.96 (t, J = 1.8 Hz, 1H), 5.25 (d, J = 1.8 Hz, 1H); 13C
NMR (CDCl3, 150 MHz) δ 27.9, 30.8, 38.5, 46.5, 64.0, 82.5, 108.1,
145.2, 148.9, 175.1; All data are consistent with reported literature
values.13c
(
)-2-[(tert-Butoxy)carbonyl]-7-methylene-2-azabicyclo-
[2.2.2]octane-3-one (20). The title compound was prepared
analogously to compound 14 from 19 in 2 steps using identical
reagent stoichiometry and obtained in 71% isolated yield: Rf = 0.45
1
(50:50 EtOAc−hexanes); H NMR (CDCl3, 600 MHz) δ 1.50 (s,
9H), 1.67−1.70 (m, 1H), 1.72−1.81 (m, 1H), 1.87−1.94 (m, 2H),
2.38 (dd, J = 17.4, 2.4 Hz, 1H), 2.55 (dd, J = 17.4, 2.4 Hz, 1H), 2.65 (t,
J = 2.4 Hz, 1H), 4.77 (br s, 1H), 4.85 (br s, 1H), 4.99 (br s, 1H); 13C
NMR (CDCl3, 150 MHz) δ 22.6, 27.1, 28.0, 31.4, 41.2, 57.3, 83.0,
109.2, 143.1, 150.0, 173.8; HRMS (APCI+) calcd C13H20NO3 [M +
H]+ 238.1438, found 238.1445 (error 2.9 ppm).
(
)-Ethyl cis-(E)-3(3-{[(tert-Butoxy)carbonyl]amino}-4-
methylenecyclopentyl)acrylate (15). The title compound was
prepared analogously to compound 10 from 14 (1.0 equiv.) employing
DIBAL-H (1.5 equiv) and triethylphosphonoacetate (1.2 equiv) and
obtained in 81% isolated yield over 2 steps: Rf = 0.35 (20:80 EtOAc−
hexanes); 1H NMR (CDCl3, 600 MHz) δ 1.23−1.29 (m, 1H), 1.28 (t,
J = 7.2 Hz, 3H), 1.43 (s, 9H), 2.23−2.28 (m, 1H), 2.32−2.35 (m, 1H),
2.61−2.69 (m, 2H), 4.18 (q, J = 7.2 Hz, 2H), 4.46 (br s, 1H), 4.55 (br
s, 1H), 4.98−5.01 (m, 2H), 5.81 (d, J = 15.6 Hz, 1H), 6.88 (dd, J =
15.6, 7.2 Hz, 1H); 13C NMR (CDCl3, 150 MHz) δ 14.2, 28.3, 36.6,
38.2, 39.8, 54.5, 60.2, 79.4, 107.2, 120.6, 150.3, 150.6, 155.6, 166.5;
HRMS (APCI+) calcd for C16H26NO4 [M + H]+ 296.1856, found
296.1862 (error 2.0 ppm).
(
)-tert-Butyl cis-4-(3-hydroxypropyl)-2-methylenecyclo-
hexylcarbamate (22). To a solution of 20 (237 mg, 1.0 mmol, 1
equiv) in toluene (4 mL) at −78 °C, was added dropwise 1 M DIBAL-
H in toluene (1.5 mL, 1.5 mmol, 1.5 equiv) over 15 min. The solution
was stirred at −78 °C for 1 h and quenched by the addition of MeOH
(0.5 mL) and saturated aqueous potassium sodium tartrate (20 mL).
The mixture was gradually warmed to 25 °C and stirred for 30 min.
The aqueous layer was extracted with EtOAc (3 × 20 mL). The
combined organic layers were washed with H2O (20 mL), saturated
aqueous NaCl (20 mL), dried (MgSO4) and concentrated to afford
the desired hemiaminal intermediate, which was used directly in the
next step without further purification.
(
)-Ethyl cis-3-[3-{[(tert-Butoxy)carbonyl]amino}-4-
To a solution of triethylphosphonoacetate (0.20 mL, 1.2 mmol, 1.2
equiv) in toluene (4 mL) at 0 °C, was added NaH (60 wt % in mineral
oil, 48 mg, 1.2 mmol, 1.2 equiv) in one portion. The mixture was
stirred at 0 °C for 30 min, then a solution of the hemiaminal
intermediate from the previous step in toluene (2 mL) was added
dropwise. The reaction was gradually warmed to 25 °C and stirred for
15 h. TLC indicated complete conversion and the reaction was
partitioned between saturated aqueous NH4Cl (20 mL) and EtOAc
(20 mL). The aqueous layer was extracted with EtOAc (3 × 20 mL).
The combined organic layers were washed with H2O (20 mL),
saturated aqueous NaCl (20 mL), dried (MgSO4) and concentrated to
afford 21, which was used directly in the next step without further
purification.
To a solution of 21 prepared above (309 mg, 1.0 mmol, 1.0 equiv),
CuCl (74 mg, 0.75 mmol, 0.75 equiv) and cyclohexene (0.4 mL, 4.0
mmol, 4.0 equiv) in MeOH (20 mL) at −78 °C, was added NaBH4
(189 mg, 5.0 mmol, 5.0 equiv). The reaction was stirred at −78 °C for
1 h. The reaction was concentrated in vacuo while the flask was still
cold. The crude product was then partitioned between saturated
aqueous NH4Cl (30 mL) and EtOAc (30 mL). The aqueous layer was
extracted with EtOAc (3 × 20 mL). The combined organic layers were
washed with saturated aqueous NaCl, dried (MgSO4) and
concentrated to afford ( )-ethyl cis-3-(4-{[(tert-butoxy)carbonyl]-
amino}-3-methylenecyclohexyl)propanoate, which was used in the
next step without further purification.
methylenecyclopentyl]propanoate (16). The title compound
was prepared analogously to compound 11 from 15 (1.0 equiv),
CuCl (0.75 equiv), NaBH4 (5.0 equiv) and cyclohexene (4.0 equiv),
and obtained in quantitative isolated yield: Rf = 0.35 (20:80 EtOAc−
hexanes); 1H NMR (CDCl3, 600 MHz) δ 0.92−1.02 (m, 1H), 1.24 (t,
J = 7.2 Hz, 3H), 1.43 (s, 9H), 1.64−1.69 (m, 2H), 1.85−1.89 (m, 1H),
1.94−2.03 (m, 1H), 2.29 (t, J = 7.2 Hz, 2H), 2.28−2.33 (m, 1H), 2.57
(dd, J = 16.8, 7.2 Hz, 1H), 4.11 (q, J = 7.2 Hz, 2H), 4.36 (br s, 1H),
4.51 (br s, 1H), 4.92 (br s, 1H), 4.95 (br s, 1H); 13C NMR (CDCl3,
150 MHz) δ 14.2, 28.3, 30.6, 32.9, 35.1, 37.2, 40.4, 54.7, 60.3, 79.2,
106.3, 151.6, 155.7, 173.4; HRMS (APCI+) calcd for C16H28NO4 [M
+ H]+ 298.2013, found 298.2010 (error 1.0 ppm).
( )-cis-3-(3-Amino-4-methylenecyclopentyl)propan-1-ol hy-
drochloride salt (M-3). The title compound was prepared
analogously to compound M-2 in 2 steps. First, 16 (1.0 equiv) was
reduced with LiAlH4 to afford ( )-tert-butyl cis-4-(3-hydroxypropyl)-
2-methylenecyclopentylcarbamate in 83% isolated yield as a colorless
foam: Rf = 0.35 (50:50 EtOAc−hexanes); 1H NMR (CDCl3, 600
MHz) δ 0.97 (q, J = 10.8 Hz, 1H), 1.35−1.41 (m, 2H), 1.43 (s, 9H),
1.52−1.57 (m, 2H), 1.81−1.96 (m, 2H), 2.29−2.31 (m, 1H), 2.56 (dd,
J = 16.8, 7.2 Hz, 1H), 3.60 (t, J = 6.6 Hz, 2H), 4.34 (br d, J = 6.6 Hz,
1H), 4.55 (br d, J = 6.6 Hz, 1H), 4.90 (br s, 1H), 4.92 (br s, 1H); 13C
NMR (CDCl3, 150 MHz) δ 28.4, 31.2, 31.8, 35.5, 37.6, 40.7, 54.8,
62.8, 79.2, 106.1, 152.0, 155.8; HRMS (APCI+) calcd for C14H26NO3
[M + H]+ 256.1907, found 256.1917 (error 3.9 ppm).
To a solution of crude ( )-ethyl cis-3-(4-{[(tert-butoxy)carbonyl]-
amino}-3-methylenecyclohexyl)propanoate prepared above (311 mg,
1.0 mmol, 1 equiv) in THF (5 mL) at 0 °C, was added LiAlH4 (57 mg,
1.5 mmol, 1.5 equiv) in one portion. The reaction was stirred at 0 °C
for 30 min and quenched by the successive dropwise addition of
MeOH (0.5 mL) and saturated aqueous potassium sodium tartrate (20
mL). The mixture was warmed to 25 °C and stirred for 30 min. The
aqueous layer was extracted with EtOAc (3 × 20 mL). The combined
organic layers were washed with H2O (20 mL), saturated NaCl
solution (20 mL), dried (MgSO4) and concentrated. Purification by
silica gel column chromatography (10:90 EtOAc−hexanes to 50:50
EtOAc−hexanes) afforded the title compound (180 mg, 67% isolated
yield in 4 steps) as a colorless foam: Rf = 0.20 (50:50 EtOAc−
hexanes); 1H NMR (CDCl3, 600 MHz) δ 1.25−1.35 (m, 2H), 1.44 (s,
( )-tert-Butyl cis-4-(3-hydroxypropyl)-2-methylenecyclopentylcar-
bamate prepared above was converted to the title compound by
treatment with 4 N HCl in dioxane in 89% isolated yield as a white
1
solid: mp 153−155 °C; H NMR (CD3OD, 600 MHz) δ 1.30 (q, J =
12.0 Hz, 1H), 1.45−1.58 (m, 4H), 1.96−2.00 (m, 1H), 2.07−2.12 (m,
1H), 2.35−2.41 (m, 1H), 2.61 (dd, J = 16.2, 6.6 Hz, 1H), 3.54 (t, J =
6.6 Hz, 2H), 3.99 (t, J = 7.8 Hz, 1H), 5.21 (br s, 2H); 13C NMR
(CDCl3, 150 MHz) δ 32.3, 32.8, 38.3, 39.5, 39.6, 54.9, 63.1, 110.9,
150.1; HRMS (ESI+) calcd for C9H18NO [M + H]+ 156.1383, found
156.1390 (error 4.5 ppm).
(
)-2-Benzyl-7-methylene-2-azabicyclo[2.2.2]octan-3-one
(19). The title compound was prepared analogously to compound 13
from 18 (1.0 equiv) employing Nysted reagent (3.0 equiv) and TiCl4
(3.0 equiv), and obtained in 84% isolated yield: Rf = 0.45 (20:80
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9H), 1.52−1.71 (m, 7H), 2.03 (dd, J = 13.2, 7.2 Hz, 1H), 2.25 (dd, J =
13.2, 7.2 Hz, 1H), 3.63 (t, J = 6.6 Hz, 2H), 4.07 (br s, 1H), 4.64 (br s,
1H), 4.73 (br s, 1H), 4.85 (br s, 1H); 13C NMR (CDCl3, 150 MHz) δ
28.2, 28.4, 29.9, 30.3, 30.9, 36.4, 38.2, 53.0, 63.1, 79.3, 108.6, 146.5,
155.2; HRMS (APCI+) calcd for C15H28NO3 [M + H]+ 270.2064,
found 270.2061 (error 1.1 ppm).
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drochloride (M-4). The title compound was prepared from 22
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BioA Enzyme Assay to Determine IC50 Values. We used an
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Inhibitors were dissolved in DMSO and dispensed (1 μL of a 100×
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DMSO concentation of 1%. A master mix of 320 nM BioD, 50 nM
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derivative,17 and 5 mM SAM in reaction buffer (100 mM Bicine, 5
mM ATP, 50 mM NaHCO3, 1 mM MgCl2, 0.1 mM PLP, 0.0025%
Igepal CA630, 1 mM TCEP, pH 8.6) was prepared, and 98 μL was
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mM stock solution in H2O) providing a final concentation of 12.5 μM.
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at 530 nm and cutoff at 530 nm. The initial velocity (v0) was
determined by the time to produce 90 nM dethiobiotin. The data were
fit to the standard four-parameter Hill equation using GraphPad Prism
(version 4.0) to provide an IC50 value.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR and 13C NMR spectra for all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Center for Drug Design, University of Minnesota, 7-215
Phillips Wangensteen Building, 516 Delaware St. SE,
Minneapolis, MN 55455. E-mail: aldri015@umn.edu. Tel:
612-625-7956.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a grant from the Bill and
Melinda Gates foundation and the Wellcome Trust through the
Grand Challenges in Global Health Initiative (to Douglas
Young, Imperial College) and the National Institutes of Health
(AI091790 to Dirk Schnappinger at Weill Cornell Medical
College). We thank Daniel Wilson for performing the
inhibition assays.
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dx.doi.org/10.1021/jo3008435 | J. Org. Chem. 2012, 77, 6051−6058