Synthesis and biological evaluation of cremastrine and an unnatural analogue

Article abstract of DOI:10.1016/j.tetlet.2012.05.009

In this Letter, we describe the first total synthesis of cremastrine, a pyrrolizidine alkaloid from Cremastra appendiculata, with anticholinergic activity as well as an unnatural analogue. The streamlined synthesis proceeds in nine steps, seven steps longest linear sequence, in 25.2% overall yield, and features novel methodology to construct the pyrrolizidine core. Biological evaluation of cremastrine and the unnatural analogue indicated that both are pan-mAChR functional antagonists.

Full text of DOI:10.1016/j.tetlet.2012.05.009

Tetrahedron Letters 53 (2012) 3577–3580
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Tetrahedron Letters
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Synthesis and biological evaluation of cremastrine and an unnatural analogue
Kristopher N. Hahn a, Olugbeminiyi O. Fadeyi a, Hyekyung P. Cho b, Craig W. Lindsley a,b,
⇑
a Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
b Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt Specialized Chemistry Center (MLPCN), Vanderbilt University Medical Center,
Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter, we describe the first total synthesis of cremastrine, a pyrrolizidine alkaloid from Cremastra
appendiculata, with anticholinergic activity as well as an unnatural analogue. The streamlined synthesis
proceeds in nine steps, seven steps longest linear sequence, in 25.2% overall yield, and features novel
methodology to construct the pyrrolizidine core. Biological evaluation of cremastrine and the unnatural
analogue indicated that both are pan-mAChR functional antagonists.
Received 14 March 2012
Revised 30 April 2012
Accepted 2 May 2012
Available online 10 May 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Pyrrolizidine
Cremastrine
Anticholinergic
Sulfinimine
Mitsunobu
Recently, Ikeda et al. reported on the isolation and characteriza-
tion of cremastrine (1), a pyrrolizidine alkaloid from the Japanese
tuber Cremastra appendiculata, a species widely used in traditional
medicine in Asia for a number of therapeutic indications.1 Prelimin-
ary biological investigation, employing a [3H]-NMS binding assay,
indicates that 1 is a moderately selective muscarinic acetylcholine
receptor subtype 3 (M3) ligand (M1 Ki = 505 nM, M2 Ki > 5000 nM
M3 Ki = 126 nM, M4 Ki = 498 nM, M5 Ki = 1220 nM) an interesting
pharmacological profile if the binding profile translated into func-
tional antagonism.1,2 As such, 1 would have the potential to treat
irritable bowel syndrome, chronic obstructive pulmonary disease
and asthma.2 Based on our lab’s interest in muscarinic drug discov-
ery3 and our recently developed methodology to rapidly construct
enantiopure idolizines, pyrrolo[1,2-a]azepines, and pyrrolo[1,2-
a]azocines,4 application of this methodology to the synthesis of
the related pyrrolizidine core seemed warranted. In this Letter, we
report the first total synthesis of cremastrine (1) employing a new
synthetic strategy, distinct from previous pyrrolizidine synthe-
ses,5–8 (Scheme 1), that afforded 1 in seven total synthetic steps
and an overall yield of 25.2% that enabled biological evaluation.
by hydrolysis to furnish the enantiopure a
-hydroxy acid 3.9,10
Treatment of acid 3 with excess tert-butyldimethylsilyl chloride,
followed by basic hydrolysis, provides coupling partner 7 in 65%
yield over three steps, without epimerization and in agreement
with literature precedent (Scheme 2).9
With 7 in hand, we then targeted the synthesis of the pyrrolizi-
dine core of 1 employing our new methodology for enantiopure
OH
4'
2'
1'
3'
O
3
9
5'
H
8
1
2
O
7
6'
6
N
5
1, cremastrine
OH
OH
H
HO
+
Efforts first focused on the synthesis of the
ylic acid 3. Conversion of -isoleucine 6 to -hydroxy carboxylic
acid 3 was accomplished following the Shin procedure wherein
the -amino acid is converted into the -hydroxy acid with reten-
tion of stereochemistry.9,10 Treatment of 6 with aqueous nitrous
a-hydroxy carbox-
N
O
D
a
2
3
a
a
NH2
O
HO
O
S
N
Br
acid leads to diazotization of the
a-amine stereocenter followed
TBSO
O
O
4
5c
6
⇑
Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 345 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
Scheme 1. Retrosynthetic analysis of cremastrine (1).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.009

3578
K. N. Hahn et al. / Tetrahedron Letters 53 (2012) 3577–3580
OH
OTBS
NH2
i) TBSCl, imid.
NaNO2
H2SO4
HO
HO
HO
ii) K2CO3, H2O
MeOH
O
O
O
6
3
7
77%
85%
Scheme 2. Synthesis of
a-hydroxy acid 3 and protected congener 7.
1) PPTS
1) NaCN, DMSO
O
O
TBAI, 80 °C
O
HO
OH
O
H
2)
O3
O
O
Br
2) DIBALH, CH2Cl2
DMSO
H
9
8
10
78%
84%
O
S
H2N
93%
CuSO4
O
O
O
S
N
H
4
Scheme 3. Two approaches for the synthesis of sulfinamide 4.
homoallylic sulfinamide 11c with yields comparable to small-scale
reactions.
Table 1
In(0) allylations of sulfinamide (4) with diverse allyl bromides
Hydroboration–oxidation of the terminal olefin of 11c furnished
primary alcohol 12 in excellent yield. Initial attempts to form the
substituted pyrrolidine ring system involved mesylation of the
alcohol and displacement in a 5-exo-tet cyclization fashion. This
was unfortunately unsuccessful, but under Mitsunobu conditions,
we successfully obtained substituted pyrrolidine 13. NOESY-NMR
data confirm the syn-stereochemical relationship of the two chiral
centers on the pyrrolidine ring. From here, TBAF deprotection of
the tert-butyldimethylsilyl ether gave primary alcohol 14. Depro-
tection of the acetal and the sulfinyl exposed the aldehyde and
amine, respectively. This was theorized to undergo an intramolec-
ular condensation to form the imine, upon which an addition of a
reducing agent would produce 2,11–13 the pyrrolizidine core struc-
ture. Both classical conditions,11–13 and a variety of acids (TFA, HCl,
HOAc) and reducing agents (Et3SiH, NaBH(OAc)3, NaBH3CN) failed
to provide the desired product 2 (Scheme 4).
R
Br
O
O
H
O
S
NH
O
S
N
5a-e
Indium (0)
sat. NaBr
rt, 48 h
O
O
R
4
11a-e
Entry
R
Bromide
Sulfinamide
Yield (%)
dr
1
2
3
4
5
CO2Me
CO2Et
CH2OTBS
CH2OPMB
CH2OH
5a
5b
5c
5d
5e
11a
11b
11c
11d
11e
55
50
85
0
>6:1
>6:1
4:1
—
31
3:1
azacine construction employing Ellman sulfinimine technology via
4.4,11 Requisite aldehyde 10 is commercially available, but we also
developed two complimentary routes to access the key aldehyde
precursor 10 to sulfinamide 4 (Scheme 3). 4-Pentenal 8 was pro-
tected as the 1,3-dioxane, followed by oxonolysis to provide 10
in 78% yield. Alternatively, bromide 9 was converted into the ni-
trile congener, and reduced with DIBALH to deliver 10 in 84% yield.
Condensation of commercially available (R)-tert-butanesulfina-
mide and aldehyde 10 gives chiral aldimine 4 in 93% yield.
From aldimine 4, we next sought to install two stereocenters
through an indium mediated allylation using an appropriately
functionalized allyl bromides (Table 1).12 Interestingly, esters and
free hydroxyl moieties afforded poor conversion (30–55%), but
good dr (3:1 to >6:1). Ultimately, good yield and moderate diaste-
reoselectivity were observed when TBS–ether 5 was used provid-
ing 11 (85% conversion, >4:1 dr), and the stereochemistry
assigned based on the established literature precedent of a six-
membered ring chelation control model.13,14 For large scale prepa-
rations, we found that stirring and sonication of the indium metal
and bromide before adding the aldimine were crucial to deliver
Due to the failure of this key transformation, we presumed that
the presence of the free hydroxyl in the protic environment led to
undesired side reactions leading to very polar, complex mixtures.
Therefore, we modified our approach to cyclize after formation of
the ester bond. As shown in Scheme 5, an EDCI/DMAP mediated
coupling between acid 7 and alcohol 14 delivered ester 15 in excel-
lent yield. Application of the classical deprotection/reductive ami-
nation conditions (TFA–H2O (95:5) followed by triethyl silane as
the reducing agent afforded both global deprotection of 15 as well
as intramolecular condensation/reductive amination to furnish the
natural product cremastrine (1) in 80% yield from ester 15. Our
synthetic 1 was in agreement with the data reported for natural
1;15 however, only tabular NMR data are available and authentic
samples could not be acquired. Thus, the first total synthesis of 1
has been completed in seven steps (five steps longest linear se-
quence) with an overall yield of 25.2% from commercial starting
materials. This expedited route also allowed for the preparation
of quantities of 1 to support biological studies.
Following the route in Scheme 2, we also prepared a benzyl pro-
tected congener 16 of 7, to explore structure–activity relationships

K. N. Hahn et al. / Tetrahedron Letters 53 (2012) 3577–3580
3579
O
O
O
S
NH
O
S
NH
9-BBN, H2O2
DEAD, PPh3
O
NaOH, CH2Cl2
95%
O
THF
72%
OTBS
OTBS
HO
11c
12
O
O
OTBS
OH
O
TFA:H2O (95:5)
Et3SiH
O
TBAF, THF
93%
O
N
2
O
N
S
S
13
13
Scheme 4. Attempted synthesis of pyrrolidine core 2.
OTBS
O
O
O
OH
7
, EDCI, DMAP
CH2Cl2
O
O
O
N
N
O
O
96%
S
S
14
15
OH
O
OH
H
N
O
H
N
TFA:H2O (95:5)
Et3SiH
80%
O
O
cremastrine (1)
Scheme 5. Completion of the synthesis of cremastrine (1).
OBn
IC50 >10 lM, M5 IC50 = 4.0 lM), with a preference for M1 and M5,
HO
and very weak activity at M3. Thus, 1 is not a selective M3 antago-
nist. Unnatural analogue 17 was more potent and selective than 1
O
O
OH
16
OBn
(M1 IC50 = 1.9
lM, M2 IC50 >10
lM, M3 IC50 = 5.0 lM, M4
O
1. EDCI, DMAP
O
H
N
IC50 = 5.2 M, M5 IC50 = 2.0
l
l
M), but still categorized as a pan-
CH2Cl2
O
N
mAChR antagonist. These data suggest that the azabicy-
clo[3.3.0]octane ester chemotype is comparable to the pan-mAChR
antagonist tropane ester chemotypes, such as atropine.18 However,
these data are still intriguing and the synthesis and evaluation of
additional unnatural analogues would be of scientific value.
In summary, we have successfully extended our methodology
to rapidly construct enantiopure idolizidines, pyrrolo[1,2-a]aze-
pines and pyrrolo[1,2-a]azocines,4 to the synthesis of pyrrolizidine
alkaloids. Here, we completed the first total synthesis of the pyrr-
olizidine alkaloid cremastrine (1) in seven steps from commercial
materials and in 25.2% overall yield. The methodology also allowed
for the synthesis of unnatural analogue such as 17. Moreover, we
demonstrated in mAChR functional assays, that both 1 and unnat-
ural 17 are not selective M3 antagonists, as previous binding data
suggested, but rather pan-mAChR antagonists. This concise synthe-
sis will allow for the synthesis of additional unnatural analogues of
1 for biological evaluation as well as for the synthesis of related
pyrrolizidine-based alkaloid natural products. These efforts are in
progress and will be reported in due course.
O
S
2. TFA:H2O (95:5)
3.
Et3SiH
77.6% over 3 steps
Scheme 6. Synthesis of unnatural analogue 17.
17
14
(SAR) with a non-hydrolyzable protecting group in place of the free
hydroxyl of 1. As shown in Scheme 6, unnatural analogue 17 was
readily prepared and once again in high overall yield (26.1%).16
Unnatural analogue 17 also provided confidence for the synthesis
of 1 (since we were unable to acquire NMRs or an authentic sam-
ple), as spectral data for 17 agreed well with synthetic and natural
1.
As cremastrine (1) was reported to display modest selectivity
for binding to the M3 mAChR (2- to 20-fold),1 we evaluated both
our synthetic 1 and unnatural analogue 17 against all five human
mAChRs (M1–M5) in a functional assay to determine if the mod-
estly selective M3 binding would translate into selective M3 func-
tional antagonist activity.17 Interestingly, we found that 1 was a
pan-mAChR functional antagonist (Fig. 1), moderately inhibiting
Acknowledgments
The authors gratefully acknowledge funding from the Depart-
ment of Pharmacology, Vanderbilt University Medical Center and
M1–M5 (M1 IC50 = 2.8 lM, M2 IC50 >10 lM, M3 IC50 >10 lM, M4

3580
K. N. Hahn et al. / Tetrahedron Letters 53 (2012) 3577–3580
Figure 1. Human mAChR (M1–M5) functional assay with cremastrine 1 and unnatural analogue 17, indicating that both are functional antagonists of all five mAChRs.
(19 mg, 80% yield) as a colorless oil. ½a D20
ꢀ
ꢁ15.2 (c 0.97 CHCl3; ½a D20
ꢁ27.2 (c 1.0,
ꢀ
the NIH/Molecular Libraries Production Center Network (MLPCN)
(U54MH084659).
EtOH); 1H NMR (400.1 MHz, CDCl3) d (ppm) = 6.87 (m, 1H), 4.49 (d, J = 5.2, 1H),
4.33 (m, 1H), 4.20 (m, 1H), 4.18 (m, 1H), 3.55 (m, 1H), 3.49 (t, J = 7 Hz 1H), 2.88
(m, 1H), 2.8 (m, 1H), 2.65 (dt, J = 8.1, 1H), 1.99 (m, 1H), 1.90–1.80 (br m, 4H),
1.72 (m, 2H), 1.59 (m, 1H), 1.42 (m, 1H), 1.1 (d, J = 4 Hz, 3H), 0.99 (t, J = 7 Hz,
3H). 13C NMR (100.6 MHz, pyr-d5) d (ppm): 174.6, 72.6, 68.1, 63.1, 55.38, 53.27,
39.9, 39.46, 26.64, 26.2, 25.93, 24.78, 15.81, 11.75; HRMS (TOF, ES+) C14H26NO3
[M+H]+ calcd 256.1913, found 256. 1911.
References and notes
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2. Peretto, I.; Petrillo, P.; Imbimbo, B. P. Med. Res. Rev. 2009, 29, 867–902.
3. Conn, P. J.; Jones, C.; Lindsley, C. W. Trends Pharm. Sci. 2009, 30, 148–156.
4. (a) Fadeyi, O. O.; Senter, T. J.; Hahn, K. N.; Lindsley, C. W. Chem. Eur. J. 2012, 18,
5826–5831; (b) Senter, T. J.; Fadeyi, O. O.; Lindsley, C. W. Org. Lett. 2012, 14,
1869–1871.
5. Vanecko, J. A.; West, F. G. Org. Lett. 2005, 7, 2949–2952.
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7. Fürstner, A.; Kortte, A. Chem. Asian J. 2008, 3, 310–318.
16. A solution of ester (50 mg, 0.0914 mmol) in 2 mL of 95:5 trifluoroacetic acid–
H2O was stirred for 1 h at room temperature then 0.19 mL of triethyl silane was
added. This solution was stirred overnight and concentrated in vacuo to give a
crude, yellow oil. This was dissolved in 1 mL DMSO and purified by reverse
phase chromatography (2% to 45% H2O (0.1% TFA)–AcCN) to give (19 mg, 80%
yield) as a colorless oil. ½a D20
ꢀ
(c 1.12, CHCl3); 1H NMR (400.1 MHz, CDCl3) d
(ppm) = 7.33 (m, 5H), 4.66 (d, J = 11.49, 1H), 4.38 (d, J = 11.50, 1H) 4.15 (dt,
J = 11.26, 4.89 Hz, 2H), 3.74 (d, J = 5.65 Hz, 1H), 3.67 (m, 1H), 3.46 (m, 1H),
3.11–2.92 (m, 3H), 2.41 (sextet, J = 6.77 Hz, 1H), 2.01 (m, 1H), 1.88–1.78 (m,
3H), 1.58 (m, 2H), 1.25 (m, 3H), 0.91 (d, J = 6.77 Hz, 3H), 0.87 (t, J = 7.91, 3H);
13C NMR (100.6 CDCl3) d (ppm): 172.44, 137.48, 128.37, 127.86, 82.65, 72.53,
64.69, 62.72, 61.18, 44.29, 41.49, 38.00, 31.30, 29.68, 28.75, 28.05, 24.71, 15.17,
11.28; HRMS (TOF, ES+) 346.2382, found 346.2381.
8. Ishibashi, H.; Sasaki, M.; Taniguchi, T. Tetrahedron 2008, 64, 7771–7773.
9. Shin, M. R.; Lee, J.; Lee, M.; Jung, W.; Lee, J.; Yoon J. Org. Chem. 2000, 65, 7667–
7675.
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12. Sun, X.-W.; Liu, M.; Xu, M.-H.; Lin, G.-Q. Org. Lett. 2008, 10, 1259–1262.
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14. Robak, M. T.; Herbage, M. A.; Ellman, J. A. Chem. Rev. 2010, 110, 3600–3740.
15. A solution of ester 15 (50 mg, 0.0914 mmol) in 2 mL of 95:5 trifluoroacetic
acid–H2O was stirred for 1 h at room temperature then 0.19 mL of triethyl
silane was added. This solution was stirred overnight and concentrated in
vacuo to give a crude, yellow oil. This was dissolved in 1 mL DMSO and purified
by reverse phase chromatography (2% to 45% H2O (0.1% TFA)–AcCN) to give
17. For full details of the mAChR functional assay see: Lebois, E. P.; Bridges, T. M.;
Dawson, E. S.; Kennedy, J. P.; Xiang, Z.; Jadhav, S. B.; Yin, H.; Meiler, J.; Jones, C.
K.; Conn, P. J.; Weaver, C. D.; Lindsley, C. W. ACS Chem. Neurosci. 2010, 1, 104–
121.
18. Schulte, M. L.; Lindsley, C. W. Bioactive Heterocyclic Compound Classes
Pharmaceuticals and Agrochemicals In Lamberth, N., Dinges, J., Eds.; Wiley-
VCH Verlag GmbH & Co., 2012; pp 23–36.