Development of an automated microfluidic reaction platform for multidimensional screening: Reaction discovery employing bicyclo[3.2.1]octanoid scaffolds

Article abstract of DOI:10.1021/jo901073v

(Chemical Equation Presented) An automated, silicon-based microreactor system has been developed for rapid, low-volume, multidimensional reaction screening. Use of the microfluidic platform to identify transformations of densely functionalized bicyclo[3.2

Full text of DOI:10.1021/jo901073v

pubs.acs.org/joc
Development of an Automated Microfluidic Reaction Platform for
Multidimensional Screening: Reaction Discovery Employing
Bicyclo[3.2.1]octanoid Scaffolds
John R. Goodell,‡ Jonathan P. McMullen,† Nikolay Zaborenko,† Jason R. Maloney,‡
Chuan-Xing Ho,‡ Klavs F. Jensen,*,† John A. Porco Jr.,*,‡ and Aaron B. Beeler*,‡
‡Department of Chemistry and Center for Chemical Methodology and Library Development (CMLD-BU),
Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, and †Department of Chemical
Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 66-342, Cambridge,
Massachusetts 02139.
beelera@bu.edu
Received May 21, 2009
An automated, silicon-based microreactor system has been developed for rapid, low-volume,
multidimensional reaction screening. Use of the microfluidic platform to identify transformations
of densely functionalized bicyclo[3.2.1]octanoid scaffolds will be described.
Introduction
the decreasing availability of new chemical entities for bio-
logical screening and the need for discovery of new synthetic
methodologies to access novel compounds of interest.4
Miniaturization and automation of biological screening
have enabled an unprecedented level of throughput.5 How-
ever, such developments have been slower in the area of
chemical reaction discovery.6 The recent rise of microfluidic
technologies has presented an opportunity for the develop-
ment of tools to enable small-scale, rapid chemical reactions
which may be applied to discovery endeavors.7 Herein, we
Historically, major pharmaceutical breakthroughs have
been enabled by complex chemical structures;often, com-
plex natural products;with unique biological mechanisms.1
Recently, there have been numerous efforts to discover new
complex chemical scaffolds through organic synthesis.2 As
part of our interest in the synthesis of new chemotypes and
structural frameworks, we recently formulated a new ap-
proach to chemical reaction discovery termed “multidimen-
sional reaction screening”.3 In this approach, chemical
reactions are evaluated by using multiple variables in an
array format. Efforts such as multidimensional reaction
screening address an ongoing challenge in drug discovery:
(4) For discussion of reaction screening approaches, see: (a) Weber, L.;
Illgen, K.; Almstetter, M. Synlett 1999, 366. (b) Kana, W. M.; Rosenman, M.
M.; Sakurai, K.; Snyder, T. M.; Liu, D. R. Nature 2004, 431, 545. (c) Miller,
S. J. Nat. Biotechnol. 2004, 22, 1378.
(5) Liu, B.; Li, S.; Hu, J. Am. J. PharmacoGenomics 2004, 4, 263.
(6) For examples of automated screening of reaction conditions, see: (a)
Harre, M.; Neh, H.; Schulz, C.; Tilstam, U.; Wessa, T.; Weinmann, H. Org.
Process Res. Dev. 2001, 5, 335. (b) von Wangelin, A. J.; Neumann, H.;
Gordes, D.; Klaus, S.; Jiao, H.; Spannenberg, A.; Kruger, T.; Wendler, C.;
Thurow, K.; Stoll, N.; Beller, M. Chem.;Eur. J. 2003, 9, 2273. (c) Di, L.;
McConnell, O. J.; Kerns, E. H.; Sutherland, A. G. J. Chromatogr., B 2004,
809, 231. (d) Dinter, C.; Weinmann, H.; Merten, C.; Schutz, A.; Blume, T.;
Sander, M.; Harre, M.; Neh, H. Org. Process Res. Dev. 2004, 8, 482. (e)
Rudolph, J.; Lormann, M.; Bolm, C.; Dahmen, S. Adv. Synth. Catal. 2005,
347, 1361.
(1) (a) Balamurugan, R.; Dekker, F. J.; Waldmann, H. Mol. BioSyst.
2005, 1, 36. (b) Arve, L.; Voigt, T.; Waldmann, H. QSAR Comb. Sci. 2006,
25, 449.
(2) (a) Schreiber, S. L. Science 2000, 287, 1964. (b) Tan, D. S. Nat. Chem.
Biol. 2005, 1, 74. (c) Shang, S.; Tan, D. S. Curr. Opin. Chem. Biol. 2005, 9, 248.
(d) Wipf, P.; Coleman, C. M.; Janjic, J. M.; Iyer, P. S.; Fodor, M. D.; Shafer,
Y. A.; Stephenson, C. R. J.; Kendall, C.; Day, B. W. J. Comb. Chem. 2005, 7,
322. (e) Wipf, P.; Werner, S.; Woo, G. H. C.; Stephenson, C. R. J.; Walczak,
M. A. A.; Coleman, C. M.; Twining, L. A. Tetrahedron 2005, 61, 11488. (f)
Thomas, G. L.; Wyatt, E. E.; Spring, D. R. Curr. Opin. Drug Discovery Dev.
2006, 9, 700. (g) Spandl, R. J.; Bender, A.; Spring, D. R. Org. Biomol. Chem.
2008, 6, 1149.
(7) For recent reviews on advancements in microfluidics, see: (a) Jensen,
K. F. Chem. Eng. Sci. 2001, 56, 293. (b) Weigl, B. H.; Bardell, R. L.; Cabrera,
C. R. Adv. Drug Delivery Rev. 2003, 55, 349. (c) Pihl, J.; Karlsson, M.; Chiu,
D. T. Drug Discovery Today 2005, 10, 1377.
(3) Beeler, A. B.; Su, S.; Singleton, C. A.; Porco, J. A Jr. J. Am. Chem. Soc.
2007, 129, 1413.
DOI: 10.1021/jo901073v
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Published on Web 07/22/2009
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FIGURE 1. Diverse functionality of the bicyclo[3.2.1]octanoid
scaffold for multidimensional reaction screening.
report the development of an automated microfluidic plat-
form and a series of multidimensional reaction screens
involving bicyclo[3.2.1]octanoid scaffolds,8 multifunctional
chemotypes9 presenting an array of functionality for reac-
tion discovery (Figure 1).
Numerous reports have indicated that reactions per-
formed within microreactors generate products in greater
yield and purity and in shorter times in comparison to
traditional batch conditions owing to the high heat and mass
transfer and capability of obtaining wider ranges of tem-
perature.10 The continuous flow operations and small vo-
lume inherent to microreactors enable multidimensional
screening to be performed quickly while minimizing required
amounts of reagents. These features also render microreac-
tors as powerful tools for screening applications involving
expensive catalysts11 and reagents that are difficult to synthe-
size in bulk.12 Additionally, microreactors are ideal for
generation of highly reactive intermediates13 and for analyz-
ing reaction conditions that cannot be easily achieved with
conventional laboratory equipment.14
FIGURE 2. Automated microfluidic platform for reaction screen-
ing. Components include the following: (a) computer with operating
software, (b) multihead syringe pump, (c) liquid handler, (d) 96-well
reagent block, (e) compression packaging that houses microreactor
and thermoelectric module, (f) UV detection for optical triggering,
(g) sampling valve, (h) 96-well plate fraction collector, and feedback
control loops (i-k) for reagent injection, temperature control, and
sample collection, respectively.
Ismagilov and co-workers recently reported studies that
highlight the advantages of microfluidic screening as an
alternative to the more commonly used 96-well plates.15,16
In their study, reactions were preloaded into capillary
cartridges as nanoliter plugs (droplets), demonstrating the
potential for microfluidic reaction screening. Continuous
flow screening conducted by Ley and co-workers17 repre-
sents another approach that offers many of the same ad-
vantages of microfluidics. In the current work, we expand
the field of reaction screening by introducing an auto-
mated microfluidic system capable of selecting chemical
reagents and solvents, as well as varying reaction times and
temperature.
FIGURE 3. Reagent storage block (left) and illustration of inert
handling in sample syringes (right).
Results and Discussion
Automated Platform. We have constructed an automated
microfluidic platform capable of conducting programmed
multidimensional arrays in a sequential format (Figure 2).
The ability to perform a series of specified reactions was
made possible by the incorporation of a liquid handler
equipped with four independently controlled sample probes.
To maintain an inert environment for reagents, we designed
and fabricated a 96-well reagent block that accommodates
commercially available glass sleeves that are sealed with a
silicon rubber septum (Figure 3). The integrity of the
reagents within the block is maintained by an inert gas
chamber mounted over the sample wells. This chamber also
serves as a source of inert gas during reagent transfer. During
the screening procedure, reagents are drawn from this re-
agent block by syringes and injected into the microfluidic
platform as boluses.
€
(8) Buchi, G; Mak, C.-P. J. Am. Chem. Soc. 1977, 99, 8073.
(9) For examples of multifunctional scaffolds, see: (a) Couladouros, E.
A.; Strongilos, A. T. Angew. Chem., Int. Ed. 2002, 41, 3677. (b) Kumagai, N.;
Muncipinto, G.; Schreiber, S. L. Angew. Chem., Int. Ed. 2006, 45, 3635. (c)
Comer, E.; Rohan, E.; Deng, L.; Porco, J. A. Jr. Org. Lett. 2007, 9, 2123.
(10) For recent reviews on microfluidic reactions, see: (a) Fletcher, P. D.
I.; Haswell, S. J.; Pombo-Villar, E.; Warrington, B. H.; Watts, P.; Wong, S.
Y. F.; Zhang, X. Tetrahedron 2002, 58, 4735. (b) Hessel, V.; Lowe, H. Chem.
Eng. Technol. 2005, 28, 267. (c) Watts, P.; Haswell, S. J. Chem. Soc. Rev.
2005, 34, 235. (d) Geyer, K.; Codee, J. D. C.; Seeberger, P. H. Chem.;Eur. J.
2006, 12, 8434. (e) Watts, P.; Wiles, C. Chem. Commun. 2007, 443.
(11) Allwardt, A.; Holzmuller-Laue, S.; Wendler, C.; Stoll, N. Catal.
Today 2008, 137, 11.
(12) Yoshida, J.; Nagaki, A.; Yamda, T. Chem.;Eur. J. 2008, 14, 7450.
(13) Usutani, H.; Tomida, Y.; Nagaki, A.; Okamoto, H.; Nokami, T.;
Yoshida, J. J. Am. Chem. Soc. 2007, 129, 3046.
(14) De Mas, N.; Gunther, A.; Schmidt, M. A.; Jensen, K. F. Ind. Chem.
Eng. Res. 2003, 42, 698.
(15) Chen, D. L.; Ismagilov, R. F. Curr. Opin. Chem. Biol. 2006, 10, 226.
(16) Song, H.; Chen, D. L.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2006,
45, 7336.
Pulse Flow. Our specific goal was to develop a screening
platform capable of executing one to several thousand
analytical scale reactions. One microfluidic technique utilizes
a two-phase flow system, where the reaction droplets are
(17) (a) Baxendale, I. R.; Griffiths-Jonesm, C. M.; Ley, S. V.; Tranmer,
G. K. Chem.;Eur. J. 2006, 12, 4407. (b) Baxendale, I. R.; Deeley, J.;
Grithiths-Jones, C. M.; Ley, S. V.; Saaby, S.; Tranmer, G. K. Chem Commun.
2006, 2566. (c) Baumann, M.; Baxendale, I. R.; Ley, S. V.; Smith, C. D.;
Tranmer, G. K. Org. Lett. 2006, 8, 5231.
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FIGURE 5. Layout of the silicon microreactor used for multidi-
mensional screens.
FIGURE 4. (a) Illustration of sequential reaction pluses and the
dispersion caused by laminar flow and surface wetting. (b) Example
of sample collection via optical triggering. Collection begins and
terminates once concentration of reaction pulse crosses a specified
threshold, thereby enabling collection of the central portion of the pulse.
FIGURE 6. Compression packaging unit used in screening appli-
cations. The unit houses the microreactor and thermoelectric device
used for heating/cooling.
separated by a fluorinated carrier fluid or a gas phase.15,18,19
However, both of these methods have limitations in reac-
tion screening applications. For example, solubility of the
fluorinated liquid into the reaction droplet increases with
higher temperatures,20 and the use of a gas phase may lead
to cross-contamination of the reaction droplets via the liquid
meniscus between two droplets. Therefore, a comprehensive
range of reaction conditions without cross-contamination
was obtained by injecting reaction pulses into a continuous
stream of solvent and by incorporating an optically triggered
collection system. As illustrated in Figure 4, reagent pulses
(boluses) were individually introduced into the microreactor
through the liquid handler and coalesced into a single reac-
tion pulse after entering the microreactor.21
Reactor Design. A silicon microreactor was developed
based on a design previously used for liquid-phase23 and
gas-liquid24 reaction optimization. In the current design,
use of T-junctions and increased serpentine channels results
in improved mixing over a broader range of chemical
applications compared to the previous reactor design. The
microreactor layout consists of three fluid inlets, followed by
an 18 μL mixing zone and a 69 μL reaction zone, which
terminate at the quench inlet (Figure 5). The quenched
mixture flows through another mixing zone to ensure full
and rapid quenching of the reaction.
The microfluidic channels and ports were etched into
silicon wafers by a deep reactive ion etch (DRIE). The sili-
con surface was then oxidized to silica (glass) and anodi-
cally bonded to a Pyrex wafer to cap the channels, providing
a glass surface that is compatible with most organic sol-
vents. The high thermal conductivity of silicon microreac-
tors results in fast temperature equilibration; its mecha-
nical strength enables reactions to be investigated at
pressures up to 100 bar with an appropriate packaging
technique.25
A disadvantage of applying this homogeneous flow style is
the axial dispersion of the reaction pulses associated with
laminar flow (Figure 4a).22 To account for this dispersion, an
optically triggered fraction collection system was established
to collect the central section of these pulses, while the tail
ends were discarded (Figure 4b). The dispersion was also
reduced by minimizing dead volumes and the lengths of
PTFE tubing between the injector and the reactor. Further-
more, programmed delays between the reaction pulses were
incorporated to prevent cross-contamination.
Fluidic connections to the chip were achieved with a
compression packaging scheme (Figure 6). This compression
chuck houses the microreactor and a thermoelectric (TE)
€
(18) Gunther, A.; Jensen, K. F. Lab Chip 2006, 6, 1487.
(19) Hatakeyama, T.; Chen, D. L.; Ismagilov, R. F. J. Am. Chem. Soc.
2006, 128, 2518.
(20) Gladysz, J. A.; Curran, D. P.; Horvath, I. T. E., Handbook of
Fluorous Chemistry, 1st. ed.; Wiley-VCH: New York, 2004.
(21) Garcia-Egido, E.; Spikmans, V.; Wong, S. Y. F.; Warrington, B. H.
Lab Chip 2003, 3, 73.
(23) Ratner, D. M.; Murphy, E. R.; Jhunjnunwala, M.; Snyder, D. A.;
Jensen, K. F.; Seeberger, P. H. Chem. Commun. 2005, 5, 578.
(24) Murphy, E. R.; Martinelli, J. R.; Zaborenko, N.; Buchwald, S. L.;
Jensen, K. F. Angew. Chem., Int. Ed. Engl. 2007, 10, 1734.
(25) Murphy, E. R.; Inoue, T.; Sahoo, H. R.; Zaborenko, N.; Jensen, K.
F. Lab Chip 2007, 7 1309.
€
(22) Gunther, A.; Khan, S. A.; Thalmann, M.; Trachsel, F.; Jensen, K. F.
Lab Chip 2004, 4, 278.
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TABLE 1. Synthesis of Bicyclo[3.2.1]octanoid Substratesa
third inlet flow. This coalescence was confirmed by using
FEMLAB simulations. At a flow rate of 15 μL/min (5.8 min
residence time), the simulations also showed that 90% of
the mixing occurs within the first 15% of the mixing zone
(3% of total residence time), ensuring fast mixing of the
components.
Reaction times ranging from 1 to 30 min were controlled
by adjusting the flow rate of the multihead syringe pump.
The quench stream in the reactor is an important feature that
sets finite and uniform reaction times for the sequential
reactions. Temperatures ranging from -30 to 100 °C were
achieved by using the thermoelectric module and incorpor-
ating a sufficient heat sink. Collection of individual reaction
pulses was achieved by coupling an optical trigger, an
analytical scale switch, and a 96-well plate fraction collector.
Analysis of the reaction screens was performed by UPLC/
MS/ELSD.
Scaffold Synthesis. The synthesis of bicyclo[3.2.1]octanoid
scaffolds derived from [5þ2] cycloaddition of quinone
monoketals and styrene derivatives was first reported by
€
Buchi and co-workers in their work toward the synthesis of
neolignan natural products.8 Further efforts resulted in an
improved synthesis of bicyclo[3.2.1] derivatives.28,29 On the
basis of these studies, we were able to develop optimized
conditions for the synthesis of five bicyclo[3.2.1] substrates
(Table 1) using carefully controlled addition of water. The
relative stereochemistry of bicyclo[3.2.1] scaffold 7 was con-
firmed by X-ray crystallographic analysis.30 The relative
stereochemistry of bicyclo[3.2.1] substrates 8-11 was con-
firmed via 1H coupling constants and 1D NOE analyses.30
During reaction optimization, we observed that when
rigorously water-free conditions were utilized for [5þ2]
cycloaddition of monoketal 131 and styrene (Scheme 1),
the major product was diene 12 rather than the desired
bicyclo[3.2.1]octanoid 7. Ring-opened product 12 appears
to arise from a retro-Dieckmann-type process with oxo-
nium intermediate 13 and the methanol liberated du-
ring formation of oxonium intermediate 14.32 Addition of
water effectively competes with methanol to preferen-
tially afford the desired bicyclo[3.2.1]octanoid 7. However,
the addition of >1 equiv of water resulted in competing
hydrolysis of oxonium intermediate 14 affording benzo-
quinone 15. Reaction optimization revealed that the opti-
mal amount of water needed in the reaction to maximize
the yield of the desired bicyclo[3.2.1]octanoid 7 was 0.65 to
0.75 equiv.
aConditions: alkene (2.0 equiv), dry CSA (1.0 equiv), anhydrous
CH3CN, H2O (0.75 equiv), rt, 2 h.
SCHEME 1. Synthesis of Bicyclo[3.2.1]octanoid Scaffolds
element capable of heating or cooling the microreactor
by adjusting the electric current.26,27 The wetted mate-
rial of the compression piece (top half) is made from stain-
less steel to ensure compatibility with a broad range of
reagents, while the bottom portion is constructed from
aluminum to create a sufficient heat sink with high thermal
conductivity for the thermoelectric device. The reaction
temperature is monitored with a thermoresistor that is in
direct contact with the microreactor and is inserted into the
top plate.
As previously mentioned, it is necessary for each reagent
pulse to enter the main reactor channels simultaneously.
Accomplishing this task required the compression piece
ports and the inlet channels to be designed such that the
pressure drops across each reagent inlet channel were
equal. Such an arrangement allows the first two reagents
to enter the first T-mixer simultaneously, and the resulting
stream enters the second T-mixer at the same time as the
Multidimensional Screening of Bicyclo[3.2.1]octanoid Scaf-
folds. The dense functionality of multifunctional bicyclo-
[3.2.1]octanoid scaffolds such as 7 and 9 is highly desirable
for reaction discovery. With use of these scaffolds, 23
heteroatom nucleophiles, 16 carbon/dipole nucleophiles,
and 16 electrophiles (reaction partners) were screened using
the microfluidic platform (Figure 7). Due to the presence of
multiple electrophilic centers on the substrate, reaction
(28) Collins, J. L.; Grieco, P. A.; Walker, J. K. Tetrahedron Lett. 1997, 38,
1321.
(29) For additional examples of [5þ2] cycloadditions, see: (a) Engler, T.
A.; Letavic, M. A.; Combrink, K. D.; Takusagawa, F. J. Org. Chem. 1990,
55, 5810. (b) Harmata, M.; Rashatasakhon, P. Tetrahedron 2003, 59, 2371.
(30) See the Supporting Information for complete experimental details.
(31) Clive, D. L.; Fletcher, S. P.; Liu, D. J. Org. Chem. 2004, 69, 3282.
(26) Albrecht, J.; Jensen, K. F. Electrophoresis 2006, 27, 4960.
(27) Park, J.; Park, K.; Shin, K.; Park, H.; Kim, J.; Kim, R.; Park, S.;
Song, Y. Sens. Actuators, B 2006, 117, 516.
€
(32) (a) Mak, C.-P.; Buchi, G. J. Org. Chem. 1981, 46, 1. (b) Maki, S.;
Asaba, N.; Koremura, S.; Yamamura, S. Tetrahedron Lett. 1992, 33, 4169.
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FIGURE 8. UPLC/ELSD results for successful transformations
with bicyclo[3.2.1] substrate 7. The shaded region represents the
LC retention time for bicyclo[3.2.1] substrate 7. Reaction conditions
for displayed traces: DMSO, TBD, 5 min, 30 °C.
FIGURE 7. Multidimensional screening parameters.
partners capable of tandem nucleophilic additions (e.g., 22,
23, 24, 26, and 32) were also included. Two non-nucleophilic
bases 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), were utilized in the
multidimensional screen. A complete representation of the
screening dimensions is shown in Figure 7.
Each reaction was performed with 1 μmol (typically
0.25 mg) of substrate, with 2.0 to 3.0 equiv of reaction
partner and 1.2 equiv of base with a total reaction volume
of 24 μL. The latter volume was achieved by simultaneously
delivering 8 μL of each reaction component from stock
solutions in the reagent storage block (Figure 3) to the
reactor. The required stock solutions were prepared in both
DMSO and CH3CN in oven-dried glass sleeves with minimal
exposure to air as 0.125 M solutions of the bicyclo[3.2.1]
substrates, 0.250 M solutions of the reaction partners, and
0.150 M solutions of the bases.
Reaction times of 5 and 20 min were achieved by utilizing
set flow rates of 4.5 and 1.2 μL/min, respectively (multihead
syringe pump with 4-10 mL syringes). Reactions were
quenched in the reactor with 10% v/v acetic anhydride
(continuous flow) and collected into 96-well plates by using
UV-triggered fraction collection. The diluted reactions (final
concentration = 1 mg/mL) were analyzed by using UPLC/
MS/ELSD33 without further preparation. Reactions afford-
ing >20% conversion to a major product were subsequently
scaled up and isolated by using traditional bench chemistry
methods for further characterization of reaction products.
Analysis of UPLC/MS/ELSD data for the reaction screen
indicated positive outcomes for 11 out of 23 heteroatom
FIGURE 9. UPLC/ELSD results for successful transformations
with bicyclo[3.2.1] substrate 9. The shaded area represents the LC
retention time for bicyclo[3.2.1] substrate 9. Reaction conditions for
displayed traces: CH3CN, TBD, 5 min, 30 °C.
nucleophiles (Figures 8 and 9). All primary amine nucleo-
philes afforded retro-Dieckmann-type ring-opened products
72-93 (Tables 2 and 3). Although positive results utilizing
(33) Mazzeo, J. R.; Neue, U. D.; Kele, M.; Plumb, R. S. Anal. Chem.
2005, 77, 460 A.
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TABLE 2. Results for Reactions with Bicyclo[3.2.1] Substrate 7a
aReaction conditions: nucleophile (2 equiv), DBU (1.2 equiv), anhydrous CH3CN, room temperature, 1 h. bTBD (1.2 equiv): 50% yield (8:1).
cNucleophile (12 equiv). dTBD (1.2 equiv).
TABLE 3. Results for Reactions with Bicyclo[3.2.1] Substrate 9a
aReaction conditions: nucleophile (2 equiv), TBD (1.2 equiv), anhydrous CH3CN, room temperature, 1 h. bNucleophile (12 equiv).
6174 J. Org. Chem. Vol. 74, No. 16, 2009

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bicyclo[3.2.1] substrate 7 were observed when either DBU or
TBD was used, higher rates of conversion were generally
observed by using the latter base. Interestingly, positive
results utilizing substrate 9 were only observed when TBD
was used as base.
SCHEME 2. Further Cyclization of Allyl Grignard Adducts
During scale-up and optimization of ring-opened products
such as product 72, it became apparent that epimerization of
the R-amide stereocenter was occurring, giving rise to the
isolation of a mixture of diastereomers (Tables 2 and 3). It
was also noted that greater rates of epimerization occurred with
the stronger base, TBD, which is expected given a predicted pKa
of ∼25 for the R-amide proton.34 Thus, the observed diaster-
eomeric ratios reported in Tables 2 and 3 are primarily dictated
by deprotonation kinetics, which are in part dependent on
suitable alignment of the weakly acidic proton and the amide
carbonyl. The relative stereochemistry of the major diastereo-
mer of amide 83 was confirmed by X-ray crystallographic
analysis.30 Conformational analysis followed by ab initio
(B3LYP, 6-31g*) minimization of both possible diastereomers
of amides 72, 77, and 82 indicated that the major diastereomers
are thermodynamically favored by approximately 1-2 kcal/
mol.30 These data indicate that while epimerization is under
kinetic control, a thermodynamic equilibrium may exist favor-
ing the initially formed diastereomer (cf. 72-93).
SCHEME 3. Representative Products for Isonitrile Reactions
Heteroatom nucleophiles without an alkyl primary amine
(e.g., benzyl alcohol, p-anisidine, phenol, and O-ben-
zylhydroxylamine) did not react under any of the conditions
examined in the screen. This is attributed to the lower
nucleophilicity of these reaction partners in comparison to
those containing a primary alkyl amine. Similarly, secondary
amines (e.g., N-methylbenzylamine, piperidine, and pyrro-
lidine) did not react. The only exception was the reaction
between pyrrolidine (29) and bicyclo[3.2.1] substrate 7,
which afforded tertiary amide 77.
Reaction partners with the potential for tandem nucleo-
philic additions also afforded retro-Dieckmann-type ring-
opened products, but did not undergo a second, intramole-
cular addition. As shown in Tables 2 and 3, the major
isolated products were the acetylated amines and/or alcohols
that were formed by quenching with acetic anhydride. While
tandem addition products were not observed, tethered pro-
ducts were observed with diamino reaction partners 23 and
26.30 Increasing the amount of diamine to 12 equiv effec-
tively prevented the formation of the tethered products.
Utilization of chiral diamine 26 resulted in the formation
of separable diastereomers (78/79 and 89/90). The absolute
stereochemistry of 89 was confirmed by X-ray crystallo-
graphic analysis.30 The relative stereochemistries of 78, 79,
and 90 were assigned by using NMR analysis.
with approach occurring opposite the phenyl group.35
Related additions to bicyclo[3.2.1]octanoids have been re-
ported; however, in these cases stereoselectivity was not ob-
served.36
To determine scope and limitation for this reaction, addi-
tional commercially available Grignard reagents were
investigated using traditional bench chemistry methods. As
shown in Table 4, Grignard reactions proceeded with ex-
cellent yield and afforded single diastereomers. Facial-selec-
tivity for the addition to the diosphenol ether carbonyl was
confirmed by X-ray crystallographic analysis of alcohol
100.30 As indicated in Table 4 (entry 5), only allyl magnesium
bromide underwent a second addition to the bridgehead
ketone to afford tertiary alcohol 102. The relative stereo-
chemistry of diol 102 was confirmed by 1D NOE analysis.30
Furthermore, upon mild heating 102 underwent cyclization
to afford polycyclic ketal 105 (Scheme 2).
Alcohol 100 derived from the addition of methyl Grignard
was also treated with allyl magnesium bromide to afford diol
106, which upon heating gave the polycyclic ketal 107. Of the
Grignard reagents investigated, only ethynyl Grignard
(Table 4, entry 6) resulted in the formation of two regioi-
somers. In this case, addition to the enone carbonyl was still
favored, but facially selective addition to the bridgehead
ketone did occur. To confirm the relative stereochemistry of
104, the tertiary alcohol was methylated by using standard
conditions (NaH/MeI) to afford the corresponding methyl
ether which was used to conduct 1D NOE analysis.30,37
Presumably, the smaller ethynyl functionality presents a
UPLC/MS/ELSD analysis of the carbon nucleophile mi-
crofluidics screening (cf. Figure 7) also indicated positive
outcomes for reactions with 3-methoxyphenylmagnesium
bromide (54) and isonitriles 44 and 47 (Figures 8 and 9).
Interestingly, structure elucidation of products derived from
Grignard reagent 54 indicated that addition occurred at the
enone carbonyl exclusively over the bridgehead ketone
(Table 4). The addition afforded a single diastereomer, 98,
(36) Shizuri, Y.; Okuno, Y.; Shigemori, H.; Yamamura, S. Tetrahedron
Lett. 1987, 28, 6661.
(37) For examples of O-methylation to obtain NOE correlations, see: (a)
Komoda, T.; Sugiyama, Y.; Abe, N.; Imachi, M.; Hirota, H.; Koshino, H.;
Hirota, A. Tetrahedron Lett. 2003, 44, 7417. (b) Najjar, F.; Baltas, M.;
Gorrihon, L.; Moreno, Y.; Tzedakis, T.; Vail, H.; Andre-Barres, C. Eur. J.
Org. Chem. 2003, 3335.
(34) University of Wisconsin: Bordwell pKa Table (Acidity in DMSO)
http://www.chem.wisc.edu/areas/reich/pkatable/ (accessed July 14, 2008).
(35) (a) Reetz, M. T. Angew. Chem., Int. Ed. Engl. 1984, 23, 556. (b) Ye,
J.-L.; Huang, P.-Q.; Lu, X. J. Org. Chem. 2007, 72, 35.
J. Org. Chem. Vol. 74, No. 16, 2009 6175

Goodell et al.
JOCArticle
TABLE 4. Results for Reactions with Grignard Additionsa
SCHEME 4. Condensation with an Isonitrile Ylide Affords a
Polycyclic Imine
SCHEME 5. Proposed Mechanism for the Formation of Poly-
cyclic Imine 113
of this observation, we investigated an additional nitrile ylide
using a standard batch reaction (Scheme 4).40 Structure
elucidation of the isolated product revealed formation of
the unusual polycyclic imine 113. The structure was con-
firmed by X-ray crystallographic analysis.30 Selective reduc-
tion of the imine with NaBH3CN and acetic acid afforded
114 as a single diastereomer.41
aReaction conditions: Grignard reagent (3.0 equiv), anhydrous THF,
0 °C f rt, 30 min.
A proposed mechanism for the formation of imine 113 is
shown in Scheme 5 and takes advantage of the proximity of
the diosphenol ether and bridgehead ketone on the bicyclo-
[3.2.1]octanoid scaffold. Addition of the dipole to the bridge-
head ketone generates the nitrilium zwitterion 115.42 The
enol ether may then undergo intramolecular addition to the
emerged nitrilium to afford oxonium intermediate 116. Base-
mediated proton transfer generates a new zwitterion 117 that
undergoes intramolecular cyclization to afford 113.
Finally, UPLC/MS/ELSD analyses of the electrophile
reaction partner subset did not reveal any successful trans-
formations. The lack of reactivity with the electrophiles was
unexpected given the participation of the diosphenol ether
moiety observed in the formation of polycyclic imine 113.
smaller steric profile than the other Grignard reagents inves-
tigated and thus is able to approach the bridgehead ketone
from the less hindered face bearing the R-methoxy enone.
Elucidation of the successful microfluidics reactions of
bicyclo[3.2.1] substrates 7 and 9 with isonitriles 44 and 47
revealed that a [3þ2] cycloaddition had occurred at the
bridgehead ketone38 in a 1:1 diastereomeric ratio affording
spirooxazolines 108 and 109 (Scheme 3). Diastereoselectivity
of the cycloaddition occurs through addition to the less
hindered face bearing the R-methoxy enone. These structural
assignments were supported by 1D NOE analyses
(Scheme 2).30,39 The spirooxazolines 108-111 were found
to be prone to hydrolysis, affording ring-opened formami-
des.39a Reaction products derived from p-toluenesulfonyl
methyl isocyanide (44) suffered significant decomposition
while on silica and could not be cleanly isolated.
Conclusion
A microfluidic reaction platform has been developed
and used for multidimensional reaction screening of
highly functionalized bicyclo[3.2.1]octanoid scaffolds. This
Interestingly, when an isocyanide lacking an R-acidic
proton (49) was utilized, no reaction occurred. This suggests
that the formation of an isonitrile ylide intermediate39a in the
presence of base is a critical step in this process. On the basis
(40) Nair, V.; Sethumadhaven, D.; Nair, S. M.; Viji, S.; Rath, N. P.
Tetrahedron 2002, 58, 3003.
(38) For examples of dipole additions to bridgehead ketones see: (a) Van
Leusen, D.; Van Leusen, A. M. Org. React. 2001, 57, 417. (b) Groselj, U.;
Meden, A.; Stanovnik, B.; Svete, J. Tetrahedron: Asymmetry. 2007, 18, 2365.
(39) For examples of cycloadditions with isonitriles, see: (a) Soloshonok,
V. A.; Kacharov, A. D.; Avilov, D. V.; Ishikawa, K.; Nagashima, N.;
Hayashi, T. J. Org. Chem. 1997, 62, 3470. (b) Terzidis, M.; Tsoleridis, C.
A.; Stephanidou-Stephanatou, J. Tetrahedron 2007, 63, 7828.
(41) Kiss, L.; Mangelinckx, S.; Fulop, F.; De Kiimpe, N. Org. Lett. 2007,
9, 4399.
(42) For examples of stepwise cycloaddition, see: (a) Cantillo, D.; Avalos,
D.; Babiano, R.; Cintas, P.; Jimenez, J. L.; Light, M. E.; Palacios, J. C. Org.
Lett. 2008, 10, 1079. (b) Deng, X.; Mani, N. S. Org. Lett. 2008, 10, 1307. (c)
Xie, J.; Yoshida, K.; Takasu, K.; Takemoto, Y. Tetrahedron Lett. 2008, 49,
6910. (d) Shapiro, N. D.; Toste, F. D. J. Am. Chem. Soc. 2008, 130, 9244.
6176 J. Org. Chem. Vol. 74, No. 16, 2009

Goodell et al.
JOCArticle
study represents a successful application of microfluidics
technology to reaction discovery allowing for over 1000
reactions to be screened and analyzed on an analytical
scale. Use of the microfluidic platform enabled expansion
of an interesting retro-Dieckmann-type fragmentation
affording highly functionalized cycloheptenone deriva-
tives. Furthermore, [3þ2] cycloaddition involving the
bridgehead ketone of the bicyclo[3.2.1]octanoid scaffolds
and isocyanides was observed in the reaction screen. These
dipole cycloadditions inspired the ultimate discovery of a
stepwise condensation of an isonitrile ylide to afford a
novel polycyclic imine. Finally, use of moisture-sensitive
Grignard reagents with the microfluidics platform resulted
in the discovery of chemo- and stereoselective additions
to the bicyclo[3.2.1]octanoid scaffolds, highlighting the over-
all reagent capability of the screening platform. Additional
applications of microfluidics-enabled multidimensional
reaction screening and expansion of the platform capabilities
for reaction optimization and scaleup are currently under-
way and will be reported in due course.
brine (1ꢀ), dried over sodium sulfate, filtered, and evaporated
in vacuo. The crude material was purified by flash chromatog-
raphy (SiO2, 20:80 EtOAc:petroleum ether) to afford
9 (70%, 51 mg, 0.19 mmol) as clear/white crystals; mp 135-
138 °C; 1H NMR (400 MHz, CDCl3) δ 7.17-7.10 (m, 2 H),
7.09-7.03 (m, 2 H), 6.04 (d, J= 8.3 Hz, 1 H), 4.02 (dd, J=8.3,
1.8 Hz, 1 H), 3.77 (d, J = 8.3 Hz, 1 H), 3.35 (s, 3 H), 3.20 (d,
J = 17.3 Hz, 1 H), 3.12 (dd, J = 8.3, 2.0 Hz, 1 H), 2.95 (d, J =
17.3 Hz, 1 H), 1.42 (s, 3 H); NOED (400 MHz, CDCl3) irrad. δ
6.04 (Ha) 2% enhancement at Hb, irrad. δ 3.20 (Hb) 3%
enhancement at Ha; 13C NMR (100 MHz, CDCl3) δ 200.5,
190.3, 154.7, 143.8, 140.1, 128.0, 127.5, 126.2, 124.1, 114.2,
67.5, 58.1, 55.8, 55.1, 47.2, 42.5, 28.4; IR (thin film) νmax 2962,
2923, 1762, 1685, 1608, 1458, 1218, 1141, 749 cm-1; HRMS
calcd for C17H16O3Na 291.0997, found 291.1026 (MþNa).
General Procedure for Analytical Reaction Screening
(Automated Microfluidics Platform). Stock solutions of sub-
strate (0.125 M), reaction partner (0.25 M), and base (0.15 M)
were prepared in DMSO or CH3CN (anhydrous) while
maintaining dry handling. The solutions were placed in a
custom-designed 96-well aluminum holding block fitted with
oven-dried glass sleeves. This was covered with an inert gas
chamber and sealed. The block was attached to the microflui-
dics platform and a slow steady stream of argon was passed
through the inert gas chamber. System parameters were first
set to achieve 5 min reactions (microreactor residence time) at
30 °C. Longer reaction times at elevated reaction temperatures
were achieved by decreasing the syringe pump flow rate
and increasing the power supply to the TE heating module.
Reactions were quenched on the microreactor by flowing acetic
anhydride (10% V/V in DMSO or CH3CN) into the quench
port. Individual reactions were collected into 96-well plates with
use of optical detection. Each reaction was analyzed by UPLC/
MS/ELSD (10-90% CH3CN, 2 min). Reactions affording
>20% conversion to a major product were subsequently scaled
up and isolated, using traditional bench chemistry methods, for
further characterization. A sample UPLC/ELSD profile for
substrate 7 and benzylamine (16) is provided below.
Experimental Section
(1R,5R,6R,7R)-3-methoxy-6-methyl-7-phenylbicyclo[3.2.1]-
oct-3-ene-2,8-dione (()-7. To an oven-dried vial was added
dry (1S)-(þ)-10-camphorsulfonic acid (CSA) (63 mg,
0.27 mmol) followed by anhydrous CH3CN (3 mL), water
(3.7 μL, 0.21 mmol), and β-trans-methylstyrene (70 μL,
0.54 mmol). To this mixture was added 3,4,4-trimethoxycyclo-
hexa-2,5-dienone (50 mg, 0.27 mmol) dissolved in CH3CN
(1 mL). The reaction was stirred at rt for 1.5 h, quenched with
triethylamine (38 μL), and stirred for 15 min. The solution was
diluted with sat. NaHCO3 and extracted with diethyl ether (3ꢀ).
The organic layer was washed with brine (1ꢀ), dried over
sodium sulfate, filtered, and evaporated in vacuo. The crude
material was purified by flash chromatography (SiO2, 20:80
EtOAc:petroleum ether) to afford 3 (43%, 66 mg, 0.27 mmol) as
clear/white crystals. Mp 140-142 °C; 1H NMR (400 MHz,
CDCl3) δ 7.31-7.25 (m, 2 H), 7.24-7.19 (m, 1 H), 7.05 (d,
J = 8.6 Hz, 2 H), 6.48 (d, J = 8.6 Hz, 1 H), 3.81 (dd, J = 7.0, 2.0
Hz, 1 H), 3.72 (s, 3 H), 3.20 (t, J = 6.5 Hz, 1 H), 3.05 (dd, J = 8.6,
2.1 Hz, 1 H), 2.56 (qt, J = 6.7 Hz, 1 H), 1.26 (d, J = 7.0 Hz, 3 H);
13C NMR (100 MHz, CDCl3) δ 200.2, 190.3, 154.6, 138.0, 129.1,
128.6, 127.8, 118.1, 71.1, 56.0, 54.1, 49.6, 43.2, 21.7; IR (thin
film) νmax 2962, 1761, 1684, 1606, 1456, 1363, 1245, 1223, 1153,
1103, 1036, 753, 701 cm-1; HRMS calcd for C16H16O3Na
279.0997, found 279.1022 (M þ Na).
Bicyclo[3.2.1]octanoid (()-9.
To a oven-dried vial was added dry (1S)-(þ)-10-camphorsul-
fonic acid (CSA) (63 mg, 0.27 mmol) followed by anhydrous
CH3CN (3 mL), water (3.7 μL, 0.21 mmol), and 2-methylin-
dene (73 μL, 0.54 mmol). To this mixture was added 3,4,4-
trimethoxycyclohexa-2,5-dienone (50 mg, 0.27 mmol) dis-
solved in CH3CN (1 mL). The reaction was stirred at rt for
1.5 h, quenched with triethylamine (38 μL), and stirred for 15
min. The solution was diluted with sat. NaHCO3 and extracted
with diethyl ether (3ꢀ). The organic layer was washed with
(1R,6S,7R,E)-N-Benzyl-3-methoxy-7-methyl-4-oxo-6-phenyl-
cyclohept-2-enecarboxamide (()-72. To an oven-dried 1-dram
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Goodell et al.
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vial was added 3-methoxy-6-methyl-7-phenylbicyclo[3.2.1]oct-3-
ene-2,8-dione (7) (40 mg, 0.16 mmol) and anhydrous CH3CN(0.90
mL) followed by the addition of benzylamine (34 μL, 0.31 mmol).
To this mixture was added DBU (28 μL, 0.19 mmol) in anhydrous
CH3CN (100 μL). The reaction was stirred at rt for 60 min under
argon and quenched with the addition of acetic anhydride (44 μL,
0.47 mmol) in CH3CN (400 μL). The reaction was filtered through
a short plug of silica and washed with CH3CN (3 ꢀ 2 mL, until all
the product was eluted from the silica plug as indicated by TLC).
The eluent was then evaporated in vacuo. The crude material was
purified by flash chromatography (SiO2, 40:1 CH2Cl2:MeOH) to
afford 72 as a mixture of diastereomers in 20:1 ratio (dr was
estimated from crude UPLC/ELSD) (63%, 36 mg, 0.098 mmol).
Characterization data for the major diastereomer: 1H NMR (400
MHz, CDCl3) δ 7.37-7.21 (m, 8 H), 7.17-7.12 (m, 2 H), 6.20 (d,
J = 6.1 Hz, 1 H), 5.95 (t, J = 5.3 Hz, 1 H), 4.50 (dd, J = 5.5, 4.1
Hz, 2 H), 3.73 (s, 3 H), 3.71 (ovrlp t, J= 5.7 Hz, 1 H), 3.02 (dd, J =
17.6, 11.9 Hz, 1 H), 2.80 (dd, J = 17.6, 1.2 Hz, 1 H), 2.60 (dd, J =
11.1, 9.8 Hz, 1 H), 2.26-2.14 (m, 1 H), 0.88 (d, J = 6.6 Hz, 3 H);
13C NMR (100 MHz, CDCl3) δ 197.3, 171.2, 152.8, 143.8, 137.9,
128.8, 128.7, 128.0, 127.7, 126.9, 107.6, 55.5, 48.1, 45.9, 45.0, 44.1,
43.0, 15.3; IR (thin film) νmax 3337, 2961, 2927, 1666, 1537, 1454,
1345, 1208, 1140, 701 cm-1; HRMS calcd for C23H26NO3
364.1913, found 364.1937 (M þ H).
H), 7.14 (br s, 1 H), 6.14 (d, J = 5.9 Hz, 1 H), 5.96 (t, J = 5.1 Hz,
1 H), 4.60 (dd, J = 14.5, 5.5 Hz, 1 H), 4.52 (dd, J = 14.5, 5.5 Hz,
1 H), 3.70 (s, 3 H), 3.40 (d, J = 5.9 Hz, 1 H), 3.06 (d, J = 15.6 Hz,
1 H), 2.98 (dd, J = 11.7, 2.7 Hz, 1 H), 2.77-2.68 (m, 3 H), 1.15
(s, 3 H); 13C NMR (100 MHz, CDCl3) δ 196.3, 171.3, 153.1,
143.3, 141.1, 137.9, 128.8, 128.0, 127.8, 127.4, 127.2, 124.9,
124.8, 111.9, 55.4, 51.0, 50.6, 48.7, 46.6, 44.4, 44.2, 23.4; IR
(thin film) νmax 3334, 2930, 1673, 1622, 1537, 1455, 1206, 1155,
1137, 1119, 754, 734, 700 cm-1; HRMS calcd for C24H26NO3
376.1913, found 376.1919 (M þ H).
(1R,4S,5S,6R,7R)-4-Hydroxy-3-methoxy-4-(3-methoxyphenyl)-
7-methyl-6-phenylbicyclo[3.2.1]oct-2-en-8-one (()-98. To an
oven-dried vial was added 3-methoxy-6-methyl-7-phenyl-
bicyclo[3.2.1]oct-3-ene-2,8-dione (7) (40 mg, 0.16 mmol) and
anhydrous THF (4.0 mL). The reaction mixture was cooled to
0 °C while under argon. To this mixture was added 1.0 M 3-
methoxyphenylmagnesium bromide in THF (470 μL, 0.47
mmol) dropwise via syringe over 1 min while at 0 °C. The
reaction was warmed to rt with continued stirring for 30 min.
The reaction was cooled back down to 0 °C and quenched with
the addition of water (20 μL). This mixture was warmed to rt,
diluted with water (20 mL), and extracted into CH2Cl2 (3 ꢀ 15
mL). The organic fractions were combined, washed with brine
(20 mL), dried over sodium sulfate, filtered, and evaporated in
vacuo. The crude product was purified by flash chromatography
(SiO2, 40:1 CH2Cl2:EtOAc) to afford 98 as an amorphous clear/
white solid (96%, 54 mg, 0.15 mmol). 1H NMR (400 MHz,
CDCl3) δ 7.46-7.36 (m, 4 H), 7.33-7.27 (m, 1 H), 7.21-7.15 (m,
1 H), 6.83-6.81 (m, 1 H), 6.80-6.74 (m, 2 H), 5.39 (d, J = 7.4
Hz, 1 H), 3.74 (s, 3 H), 3.64 (s, 3 H), 3.09 (dd, J = 7.0, 5.9 Hz, 1
H), 2.94 (dd, J = 7.0, 1.6 Hz, 1 H), 2.88-2.78 (m, 1 H), 2.58 (dd,
(1R,6S,7R,E)-N-(4-Methoxybenzyl)-3-methoxy-7-methyl-4-
oxo-6-phenylcyclohept-2-enecarboxamide (()-77. To an oven-
dried 1-dram vial was added 3-methoxy-6-methyl-7-
phenylbicyclo[3.2.1]oct-3-ene-2,8-dione (7) (40 mg, 0.16 mmol)
and anhydrous CH3CN (0.90 mL) followed by the addition of
pyrrolidine (26 μL, 0.31 mmol). To this mixture was added TBD
(26 mg, 0.19 mmol) in anhydrous CH3CN (100 μL). The
reaction was stirred at rt for 60 min under argon and quenched
with the addition of acetic anhydride (44 μL, 0.47 mmol) in
CH3CN (400 μL). The reaction was filtered through a short plug
of silica and washed with CH3CN (3 ꢀ 2 mL, until all the
product was eluted from the silica plug as indicated by TLC).
The eluent was then evaporated in vacuo. The crude material was
purified by flash chromatography (SiO2, 40:1 CH2Cl2:MeOH)
to afford 77 as a mixture of diastereomers in 9:1 ratio (dr was
estimated from crude UPLC/ELSD) (56%, 29 mg, 0.087 mmol).
Characterization data for the major diastereomer: 1H NMR
(400 MHz, CDCl3) δ 7.36-7.22 (m, 3 H), 7.16 (d, J = 7.4 Hz, 2
H), 6.25 (d, J = 5.9 Hz, 1 H), 3.98 (t, J = 5.5 Hz, 1 H), 3.73 (s, 3
H), 3.67-3.40 (m, 4 H), 3.07 (dd, J = 17.6, 12.5 Hz, 1 H), 2.83
(d, J = 17.6 Hz, 1 H), 2.61 (dd, J = 11.7, 9.4 Hz, 1 H), 2.31-2.18
(m, 1 H), 2.06-1.97 (m, 2 H), 1.93-1.85 (m, 2 H), 0.88 (d, J =
6.6 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 197.3, 170.1, 152.6,
144.2, 128.8, 127.6, 126.8, 109.2, 55.5, 48.1, 46.5, 46.0, 43.0, 40.3,
26.3, 24.1, 15.5; IR (thin film) νmax 2966, 1728, 1680, 1625, 1452,
1209, 1137, 733, 703 cm-1; HRMS calcd for C20H25NO3Na
350.1732, found 350.1714 (M þ Na).
J = 7.4, 1.6 Hz, 1 H), 2.09 (s, 1 H), 1.07 (d, J = 7.0 Hz, 3 H); 13
C
NMR (100 MHz, CDCl3) δ 205.7, 159.4, 155.4, 144.1, 140.6,
129.5, 128.8, 128.8, 127.2, 118.1, 113.2, 111.6, 99.5, 82.7, 63.6,
55.3, 55.1, 51.2, 50.4, 42.7, 21.5; IR (thin film) νmax 3552, 2958,
1750, 1600, 1584, 1484, 1455, 1434, 1289, 1250, 1227, 1150, 1095,
1079, 1032, 784, 701 cm-1; HRMS calcd for C23H24O4Na
387.1572, found 387.1587 (M þ Na).
(1R,2R,5R,6R,7R,8S)-2,8-Diallyl-3-methoxy-6-methyl-7-phenyl-
bicyclo[3.2.1]oct-3-ene-2,8-diol (()-102.
To an oven-dried vial was added 3-methoxy-6-methyl-7-
phenylbicyclo[3.2.1]oct-3-ene-2,8-dione (7) (40 mg, 0.16
mmol) and anhydrous THF (4.0 mL). The reaction mixture
was cooled to 0 °C while under argon. To this mixture was
added 1.0 M allylmagnesium bromide in diethyl ether
(470 μL, 0.47 mmol) dropwise via syringe over 1 min while
at 0 °C. The reaction was warmed to rt with continued
stirring for 30 min. The reaction was cooled back down to
0 °C and quenched with the addition of water (20 μL). This
mixture was warmed to rt, diluted with water (20 mL), and
extracted into CH2Cl2 (3 ꢀ 15 mL). The organic fractions
were combined, washed with brine (20 mL), dried over
sodium sulfate, filtered, and evaporated in vacuo. The crude
product was purified by flash chromatography (SiO2, 40:1
CH2Cl2:EtOAc) to afford 102 as an amorphous clear/white
solid (98%, 52 mg, 0.15 mmol). 1H NMR (400 MHz, CDCl3) δ
7.32-7.22 (m, 4 H), 7.20-7.13 (m, 1 H), 6.12-5.98 (ddt,
(1R,6S,7R,E)-N-(4-Methoxybenzyl)-3-methoxy-7-methyl-4-
oxo-6-phenylcyclohept-2-enecarboxamide (()-84. To an oven-
dried 1-dram vial was added bicyclo[3.2.1]octanoid 9 (40 mg,
0.15 mmol) and anhydrous CH3CN (0.90 mL) followed by the
addition of benzylamine (30 μL, 0.30 mmol). To this mixture
was added TBD (25 mg, 0.18 mmol) in anhydrous CH3CN (100
μL). The reaction was stirred at rt for 60 min under argon and
quenched with the addition of acetic anhydride (42 μL, 0.45
mmol) in CH3CN (400 μL). The reaction was filtered through a
short plug of silica and washed with CH3CN (3 ꢀ 2 mL, until all
the product was eluted from the silica plug as indicated by TLC).
The eluent was then evaporated in vacuo. The crude material was
purified by flash chromatography (SiO2, 40:1 CH2Cl2:MeOH)
to afford 84 as a mixture of diastereomers in 7:1 ratio (dr was
estimated from crude UPLC/ELSD) (72%, 40 mg, 0.11 mmol).
Characterization data for the major diastereomer: 1H NMR
(400 MHz, CDCl3) δ 7.44-7.30 (m, 5 H), 7.20 (d, J = 4.3 Hz, 3
6178 J. Org. Chem. Vol. 74, No. 16, 2009

Goodell et al.
JOCArticle
J = 17.2, 10.1, 7.2 Hz, 1 H), 5.91-5.77 (ddt, J = 17.4, 10.2, 7.1
Hz, 1 H), 5.25-5.03 (m, 5 H), 3.56 (s, 3 H), 3.35 (dd, J = 9.2,
5.8 Hz, 1 H), 2.82 (dd, J = 5.8, 2.0 Hz, 1 H), 2.79-2.67 (ovrlp
m, 2 H), 2.64 (d, J = 7.4 Hz, 2 H), 2.60-2.52 (m, 1 H), 2.38 (s, 1
H), 2.18 (dd, J= 7.8, 2.0 Hz, 1 H), 1.28 (s, 1 H), 1.18 (d, J=7.0
Hz, 3 H); NOED (400 MHz, CDCl3) irrad. δ 3.35 (Ha) 7%
13C NMR (100 MHz, CDCl3) δ192.0, 170.6, 156.2, 153.6, 138.0,
128.7, 128.3, 127.2, 121.2, 97.5, 70.9, 68.7, 55.4, 52.4, 51.4, 48.1,
46.6, 20.4; IR (thin film) νmax 2955, 2932, 1743, 1696, 1635, 1616,
1456, 1251, 1212, 1159, 1113, 971, 736, 701 cm-1; HRMS calcd
for C20H22NO5 356.1498, found 356.1513 (M þ H). Character-
ization data for 109: 1H NMR (400 MHz, CDCl3) δ 7.29-7.21
(m, 2H), 7.21-7.14(m, 1H), 7.11 (s, 1H), 7.02 (d, J= 7.4 Hz, 2
H), 6.28 (d, J = 8.6 Hz, 1 H), 4.67 (d, J = 1.6 Hz, 1 H), 3.72 (s, 3
H), 3.56 (s, 3H), 3.56 (ovrlp t, J = 6.45 Hz, 1 H), 3.35 (dd, J =
6.6, 2.3 Hz, 1 H), 2.81 (dd, J = 8.4, 2.1 Hz, 1 H), 2.56 (d, J = 7.0
Hz, 1 H), 1.35 (d, J = 7.0 Hz, 3 H); NOED (400 MHz, CDCl3)
irrad. δ 4.67 (Ha) 3% enhancement at Hb, 1% enhancement at
Hc; irrad. δ 2.81 (Hb) 2% enhancement at Ha; 13C NMR (100
MHz, CDCl3) δ 191.1, 169.0, 155.8, 154.5, 137.6, 128.6, 128.4,
127.2, 118.0, 97.3, 70.6, 65.1, 55.7, 52.3, 52.2, 51.5, 46.3, 20.5; IR
(thin film) νmax 2955, 2932, 1745, 1692, 1632, 1615, 1244, 1207,
1176, 1162, 1138, 1103, 1009, 971, 734, 702 cm-1; HRMS calcd
for C20H22NO5 356.1498, found 356.1522 (M þ H).
13C NMR (100
00
;
0
enhancement at Hb , 5% enhancement at Hb
MHz, CDCl3) δ 160.0, 141.5, 134.7, 134.0, 128.7, 126.1, 118.5,
118.1, 99.8, 81.5, 77.6, 55.0, 53.2, 51.4, 49.1,
43.8, 43.4, 42.6, 20.6; IR (thin film) νmax 3566, 2954, 2928,
1638, 1450, 1373, 1223, 1153, 1065, 1033, 999, 912, 697 cm-1
;
HRMS calcd for C22H28O3Na 363.1936, found 363.1942
(M þ Na).
Polycyclic Ketal (()-105. To a 1-dram vial was added diol 102
(20 mg, 0.059 mmol) followed by the addition of THF (1 mL).
The reaction mixture was heated to 50 °C for 8 h while stirring.
The reaction was concentrated and purified by flash chroma-
tography (SiO2, 40:1 CH2Cl2:EtOAc) to afford 105 as an
amorphous clear/white solid (96%, 19 mg, 0.056 mmol). 1H
NMR (400 MHz, CDCl3) δ 7.31-7.27 (m, 4 H), 7.21-7.14 (m, 1
H), 6.02-5.89 (m, 1 H), 5.61-5.48 (m, 1 H), 5.20-5.12 (m, 2 H),
4.98-4.89 (m, 2 H), 3.45 (s, 3 H), 2.91 (dd, J = 9.6, 5.7 Hz, 1 H),
2.83-2.71 (m, 1 H), 2.59 (d, J = 7.0 Hz, 2 H), 2.48 (dd, J = 5.7,
1.6 Hz, 1 H), 2.32-2.14 (ovrlp m, 3 H), 2.07-1.96 (ovrlp m, 2
H), 1.24 (s, 1 H), 1.14 (d, J = 7.0 Hz, 3 H); 13C NMR (100 MHz,
CDCl3) δ 140.0, 133.6, 133.4, 128.2, 128.1, 125.9, 119.7, 117.7,
109.7, 89.1, 81.4, 56.0, 52.5, 50.9, 49.5, 41.6, 40.6, 38.1, 36.2,
22.3; IR (thin film) νmax 2953, 1498, 1456, 1447, 1311, 1217,
1154, 1134, 1096, 1064, 1032, 994, 912, 697 cm-1; HRMS calcd
for C22H28O3Na 363.1936, found 363.1944 (M þ Na).
(1R,40S,5R,50R,6R,7R)-Methyl 3-Methoxy-7-methyl-4-oxo-
6-phenyl-40H-spiro[bicyclo[3.2.1]oct[2]ene-8,50-oxazole]-40-car-
boxylate (()-108 and (1R,40R,5R,50R,6R,7R)-Methyl 3-Meth-
oxy-7-methyl-4-oxo-6-phenyl-40H-spiro[bicyclo[3.2.1]oct[2]ene-
8,50-oxazole]-40-carboxylate (()-109.
Polycyclic Imine (()-113. To a oven-dried 1-dram vial was
added N-(4-nitrobenzyl)benzimidoyl chloride (112) (40 mg,
0.15 mmol), 3-methoxy-6-methyl-7-phenylbicyclo[3.2.1]oct-3-
ene-2,8-dione (7) (25 mg, 0.097 mmol), and anhydrous toluene
(2 mL) followed by the addition of triethylamine (30.0 μL, 0.215
mmol). The reaction was stirred at rt for 24 h while under argon.
The reaction was then evaporated in vacuo and purified by flash
chromatography (SiO2, 40:1 CH2Cl2:MeOH) to afford imine
113 as a pale yellow solid (44%, 21 mg, 0.043 mmol). Mp 210-
215 °C dec; 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 9.0 Hz, 2
H), 7.98 (d, J = 7.0 Hz, 2 H), 7.85 (d, J = 9.0 Hz, 2 H), 7.64-
7.48 (m, 3 H), 7.40-7.28 (m, 3 H), 7.22 (d, J = 7.4 Hz, 2 H), 3.81
(s, 1 H), 3.52 (dd, J = 10.6, 5.1 Hz, 1 H), 3.41 (s, 3 H), 2.70 (dd,
J = 5.1, 2.3 Hz, 1 H), 2.42 (s, 1 H), 2.33 (ddd, J = 10.3, 6.9, 2.7
Hz, 1 H), 1.89 (t, J = 2.3 Hz, 1 H), 1.26 (d, J = 7.0 Hz, 3 H); 13
C
NMR (100 MHz, CDCl3) δ 207.9, 177.0, 147.5, 142.1, 136.8,
132.2, 130.5, 129.8, 129.2, 128.4, 128.2, 127.6, 127.3, 123.4,
103.2, 90.8, 88.4, 61.4, 57.4, 56.2, 56.1, 54.1, 40.9, 20.5; IR
(thin film) νmax 2959, 1750, 1603, 1518, 1497, 1448, 1349, 1271,
1151, 910, 853, 732, 697 cm-1; HRMS calcd for C30H27N2O5
495.1920, found 495.1915 (M þ H).
Polycyclic Amine (()-114.
To an oven-dried 1-dram vial was added 3-methoxy-6-methyl-7-
phenylbicyclo[3.2.1]oct-3-ene-2,8-dione (7) (40 mg, 0.16 mmol)
and anhydrous CH3CN (0.90 mL) followed by the addition
of isocyanoacetate (28.3 μL, 0.312 mmol). To this mixture
was added DBU (28 μL, 0.19 mmol) in anhydrous CH3CN
(100 μL). The reaction was stirred at rt for 60 min under
argon, quenched by filtering through a short plug of silica,
and washed with CH3CN (3 ꢀ 2 mL, until all the product was
eluted from the silica plug as indicated by TLC). The eluent was
then evaporated in vacuo. The crude material was purified by
flash chromatography (SiO2, 1:1 EtOAc:petroleum ether) to
afford 108 (35%, 19 mg, 0.055 mmol) and 109 (37%, 21 mg,
0.058 mmol) as amorphous clear/white solids. Characterization
To an oven-dried 1-dram vial was added polycyclic imine 113
(30 mg, 0.061 mmol) followed by the addition of MeOH
(1 mL). Next was added NaCNBH3 (46 mg, 0.73 mmol) and
acetic acid (42 μL, 0.73 mmol). The reaction was capped and
stirred at rt for 1 h. The reaction was extracted into CH2Cl2
(10 mL) and washed with water (2 ꢀ 10 mL) then brine
(10 mL). The organic portion was dried over Na2SO4 and
evaporated in vacuo. The resulting residue was purified by
flash chromatography (SiO2, 20:1 CH2Cl2:MeOH) to afford
amine 114 as a pale yellow solid (91%, 28 mg, 0.056 mmol).
Mp 225-230 °C dec; 1H NMR (400 MHz, CDCl3) δ 8.17 (d,
J = 8.6 Hz, 2 H), 7.74 (d, J = 9.0 Hz, 2 H), 7.56 (d, J = 7.4 Hz,
2 H), 7.44 (t, J = 7.6 Hz, 1 H), 7.37-7.21 (m, 5 H), 7.14 (d, J =
7.4 Hz, 2 H), 5.10 (d, J = 2.0 Hz, 1 H), 3.80 (s, 3 H), 3.23 (dd,
1
data for 108: H NMR (400 MHz, CDCl3) δ 7.29-7.22 (m,
2 H), 7.22-7.14 (m, 1 H), 7.08-7.00 (m, 3 H), 6.26 (d, J=
8.2 Hz, 1 H), 4.48 (s, 1 H), 3.71 (s, 3 H), 3.62 (s, 3 H), 3.51 (t, J=
6.4 Hz, 1 H), 3.39 (dd, J = 6.4, 2.0 Hz, 1 H), 3.15 (dd, J = 8.2,
2.0 Hz, 1 H), 2.53 (quin, J = 6.8 Hz, 1 H), 1.35 (d, J = 7.0 Hz,
3 H); NOED (400 MHz, CDCl3) irrad. δ 4.48 (Ha) 3%
enhancement at Hb; irrad. δ 3.39 (Hb) 2% enhancement at Ha;
J. Org. Chem. Vol. 74, No. 16, 2009 6179

Goodell et al.
JOCArticle
J = 10.7, 5.3 Hz, 1 H), 2.81 (d, J = 3.1 Hz, 1 H), 2.66 (br s, 1
H), 2.39(dd, J= 5.1, 2.3 Hz, 1 H), 2.07-1.94 (m, 1 H), 1.89 (br
s, 1 H), 0.95 (d, J = 7.0 Hz, 3 H); NOED (400 MHz, CDCl3)
irrad. δ 5.10 (Ha) 3% enhancement at Mec, 5% enhancement
at Hb; irrad. δ 3.80 (Mec) 2% enhancement at Ha, 3%
enhancement at Hb; 13C NMR (100 MHz, CDCl3) δ 210.8,
147.5, 142.1, 139.2, 137.3, 129.0, 128.6, 128.3, 128.2, 127.3,
127.1, 127.0, 123.4, 95.2, 89.3, 79.7, 63.6, 61.0, 53.7, 53.0, 52.3,
50.6, 42.8, 20.3; IR (thin film) νmax 2959, 1744, 1602, 1521,
1496, 1456, 1349, 1152, 1031, 853, 754, 699 cm-1; HRMS calcd
for C30H29N2O5 497.2076, found 497.2063 (M þ H).
Dr. Emil Lobkovsky (Cornell University) for X-ray crystal
structure analyses and Professors John Snyder, James Pa-
nek, Scott Schaus, and Michael Pollastri (Boston University)
for helpful discussions. We thank the NSF-REU program
(CHE-0649114) for support of Chuan-Xing Ho (Summer,
2007), the Microsystem Technology Laboratories of MIT
for help with microreactor fabrication, and Dr. Edward R.
Murphy for assistance and advice in establishing the micro-
fluidics screening system.
Supporting Information Available: Complete experimental
procedures and compound characterization data including X-
ray crystal structure data for 7, 83, 89, 100, and 113. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Acknowledgment. This work was generously supported
by Pfizer, Inc., the NIGMS CMLD Initiative (P50
GM067041), and Merck Research Laboratories. We thank
6180 J. Org. Chem. Vol. 74, No. 16, 2009