a. whaley- towards the synthesis and biological evaluation of 2nd-generation taxoid sb-t-1216
TRANSCRIPT
Towards the Synthesis and Biological Evaluation of 2nd-Generation Taxoid SB-T-1216
A Thesis Presented by
Adele Whaley
Stony Brook University
May 2012
Abstract of the Thesis
Towards the Synthesis and Biological Evaluation of 2nd-Generation Taxoid SB-T-1216
by
Adele Whaley
Stony Brook University
May 2012
ii
Paclitaxel and docetaxel are among the most widely used chemotherapeutic agents for the
treatment of a number of different types of cancer, such as breast, ovarian and non-small cell lung
cancer. However, these taxoids do not show efficacy against drug-resistant tumors. With the
development of the β-Lactam Synthon Method (β-LSM), a series of new-generation taxoids were
prepared, which show at least 2 orders of magnitude greater activity against a number of
drugresistant cancer cell lines. The goal of this project is to prepare highly potent new generation
taxoid SB-T-1216. In order to fulfill this goal, enantiopure β-lactam was prepared via the chiral
ester enolate-imine cyclocondensation and the Staudinger [2+2] ketene-imine cycloaddition,
followed by enzymatic kinetic resolution. The former method requires the synthesis of a chiral
ester component and a trans imine component to undergo the cyclocondensation reaction. The
ester is generated through the application of Sharpless asymmetric dihydroxylation to
1phenylcyclohexanol, followed by Raney nickel dehydroxylation to confer the appropriate
chirality to the resultant chiral alcohol. Reaction of this alcohol with an acyl chloride generates the
desired chiral ester. This ester is subsequently converted to its enolate under basic conditions. The
trans imine component is generated by aldehyde-amine condensation. The Staudinger [2+2]
ketene-imine cycloaddition requires the synthesis of a ketene component and a trans imine
component. The ketene, generated by the reaction of an acyl chloride with an amine, undergoes a
[2+2] cycloaddition with the imine component. The Staudinger [2+2] reaction generates a racemic
mixture of chiral β-lactams. The two enantiomers are resolved via enzymatic kinetic resolution to
afford enantiopure β-lactam.
β-lactam is the key intermediate used for the synthesis of 2nd-generation taxoid SB-T1216, which
shows excellent activity in a variety of cancer cell lines. SB-T-1216 is obtained in the ring-opening
coupling of this enantiopure β-lactam with a modified baccatin, followed by deprotection.
Modifications of this baccatin core consist of an N,N’-dimethylcarbamoyl substituent at the C-10
position and a tert-Butyl carbamate substituent at the C-3’ position of the isoserine side chain. The
iii
synthesis of SB-T-1216 will be presented. This material will be used towards further ongoing
research efforts to understand the unique mechanism of action of this 2nd-generation taxoid.
iv
Table of Contents
§ 1.0 Introduction ............................................................................................................................ 1
§ 1.1.0 Cancer .............................................................................................................................. 1
§ 1.2 Taxol®, Taxotere® and the 2nd-Generation Taxoids ........................................................... 2
§ 1.2.1 Chemotherapy .............................................................................................................. 2
§ 1.2.2 Discovery and Approval of Taxol® and Taxotere® for Cancer Treatment .................. 2
§ 1.2.3 Paclitaxel and Docetaxel- Mechanism of Action ........................................................ 3
§ 1.2.4 Issues with the Use of Paclitaxel and Docetaxel (Supply, Specificity, and ............... 5
Ineffectiveness against MDR cancers) .................................................................................... 5
§ 1.2.5 Resolving the Issue of Supply ..................................................................................... 6
§ 1.2.6 Resolving the Issue of MDR Cancers Through the Use of 2nd-Generation Taxoids ... 7
§ 1.3 β-Lactam Synthesis ............................................................................................................. 8
§ 1.3.1 β-Lactam ...................................................................................................................... 8
§ 1.3.2 Synthesis of Enantiopure β-lactams via the Chiral Ester Enolate-Imine
Cyclocondensation .................................................................................................................. 9
§ 1.3.3 Synthesis of Chiral β-lactams via Staudinger [2+2] Ketene-Imine Cycloaddition .. 13
Followed by Enzymatic Kinetic Resolution .......................................................................... 13
§ 1.4 SB-T-1216 Synthesis ........................................................................................................ 14
§ 1.4.1 SB-T-1216 ................................................................................................................. 14
§ 1.5 Results and Discussion ......................................................................................................... 17
§ 1.5.1 Synthesis of Whitesell’s Chiral Auxiliary ..................................................................... 17
§ 1.5.2 Synthesis of β-lactam via Chiral Ester-Enolate Imine Cyclocondensation ................... 17
§ 1.5.3 Synthesis of β-lactam via Staudinger [2+2] Ketene-Imine Cycloaddition Followed by
................................................................................................................................................... 21
Enzymatic Resolution ................................................................................................................ 21
§ 1.5.4 Synthesis of SB-T-1216 ................................................................................................. 23
§ 1.6 Experimental ......................................................................................................................... 25
§ 1.7 Summary ............................................................................................................................... 37
§ 1.8 Acknowledgments ................................................................................................................ 37
§ 1.9 References ............................................................................................................................ 37
v
1
§ 1.0 Introduction
§ 1.1.0 Cancer
Cancer is the second leading cause of death in the United States, with 1 in 4 deaths currently
attributed to this devastating disease. In 2012, an estimated total of 1.6 million new cancer cases
and 572,190 deaths are projected to occur. Despite the advances that have been made with regards
to the detection and treatment of cancer, the overall incidence and death rate has remained fairly
constant. Hence, the impact of this disease continues to remain a major problem in public
healthcare, both within the United States and around the world.1 As such, it is imperative that
efficient pharmaceutical drugs are created for the treatment of various forms of cancer.
The hallmark of cancer is the unbridled proliferation of certain cells in the body, leading to the
formation of a tumor. More often than not, this uncontrollable growth is caused by dysregulation
of the cell cycle (Figure 1-1).2
Figure 1-1 (adapted from [2]). The four phases of the cell cycle, including some of the numerous molecules important
for its progression; Extracellular events, such as growth factors, induce various signal transduction cascades that begin
in the cytoplasm (outer circle) and end in the nucleus (inner circle), leading to the activation of certain transcription
factors (TFs) and subsequent processes. The ultimate goal of this cycle is mitotic proliferation. At each stage of the
cell cycle, a checkpoint serves to ensure that defective cells do not divide. Additional mechanisms for regulation also
exist.
While healthy cells have functional checkpoints within their cell cycle that regulate their
proliferation, cancerous cells are defective in this respect. More so, additional control mechanisms
are usually lost, mutated, or have alterations in their pathway.2 A common example of the former
2
case is the loss or mutation of the genes that encode for p53, a tumor suppressing protein. A
common example of the latter case is various alterations within the retinoblastoma protein
(pRb)/E2F pathway, which plays a critical role in regulating the initiation of DNA synthesis. Like
p53, pRb is an essential tumor suppressing protein. E2F is a group of genes that encodes a family
of TFs, three of which are activators. In sum, tumorous cells accumulate a number of mutations
and defective modifications that results in constitutive mitogenic signaling. Furthermore, these
cells respond abnormally to corrective, anti-mitogenic efforts. The ramification of this synergistic
interplay is the rapid, unscheduled proliferation of these cells and the subsequent formation of a
tumor.3
§ 1.2 Taxol®, Taxotere® and the 2nd-Generation Taxoids
§ 1.2.1 Chemotherapy
Traditional chemotherapeutic methods rely on the rapid proliferation of cancerous cells, reasoning
that these aggressively dividing cells are more likely to be destroyed by a cytotoxic agent than are
normal cells. However, this lack of specificity often leads to the destruction of healthy cells that
also proliferate quickly, such as the cells lining the gastrointestinal tract, skin cells, blood cells in
the bone marrow, and hair cells.4 As a result of this systemic toxicity, a number of adverse side
effects arise, such as hair loss and nausea. Nevertheless, many of these traditional cytotoxic agents
continue to remain fundamental in the treatment of various types of cancer. Of these conventional
drugs, the small molecular members of the taxane family are rather effective, with paclitaxel and
docetaxel being most popular.
§ 1.2.2 Discovery and Approval of Taxol® and Taxotere® for Cancer Treatment
Paclitaxel was first discovered as part of a National Cancer Institute program to screen the extract
of thousands of plants for anticancer activity. Years after its characterization in 1971, paclitaxel
was commercially developed by the Bristol-Myers Squibb (BMS) biopharmaceutical company and
3
sold under the trademark Taxol® (Figure 1-2, Left).5 In December of 1992, paclitaxel was approved
by the Food and Drug Administration (FDA) for the treatment of advanced ovarian cancer. Two
years later, it also received FDA approval for the treatment of metastatic breast cancer. In 1996,
docetaxel (sold under the trademark Taxotere®), a semisynthetic analog of paclitaxel, received
FDA approval for the treatment of advanced breast cancer (Figure 1-2, Right).6
HO O OH
O O
Taxol® Taxotere®
Figure 1-2. Left: Paclitaxel (sold under the trademark Taxol®) with the cyclic carbon atoms 3 and 10 labeled in red.
Modifications at the C-3 position were made possible by the Ojima group’s development of the β-lactam Synthon
Method (β-LSM). In addition, the Ojima group experimented with modifications at the C-10 position. The addition of
certain acyl groups to this carbon made the resultant compounds 1-2 orders of magnitude more potent than either of
the parent drugs (i.e. paclitaxel and docetaxel). Right: Docetaxel (sold under the trademark Taxotere®), a semisynthetic
analog of paclitaxel, is commonly used in conjunction with paclitaxel as a fairly effective means of treating several
types of cancer.
§ 1.2.3 Paclitaxel and Docetaxel- Mechanism of Action
Paclitaxel and docetaxel are categorized as microtubule-stabilizing anticancer agents.
Microtubules are polymers of tubulin subunits, and play a critical role in many vital cellular
activities, including the maintenance of shape, motility, signal transmission, and intracellular
transport.5 The best understood and most widespread microtubules are comprised of polymers that
contain α and β tubulin. Tubulin proteins are guanosine triphosphate (GTP) binding proteins. When
in their GTP bound state, these monomers polymerize into individual protofilaments of alternating
α and β tubulin. Assembled protofilaments join into a cylindrical structure that is the final
microtubule.7 While nucleation of microtubules is unfavorable, GTP bound α- and ß-tubulin
polymerize spontaneously under physiological conditions. Microtubule associated proteins
(MAPs) regulate the properties of microtubules by carrying out a diverse degree of enzymatic
activities upon them.7
O A c O H
H O O
NH O
O
O H
O
O 1 0
3 '
O O H
O A c O H
H O O
A c O
NH O O
O
O H
3 '
1 0
4
Microtubules play an essential role in cell division by forming the mitotic spindle, which
allows replicated chromosomes to segregate to opposite poles during anaphase. During prophase,
chromosomes condense and line up at the center of the cell. During metaphase, the mitotic spindle
is formed through the alignment of microtubules at kinetochores, defined regions that are
assembled along the length of centrosomes. During anaphase, each sister chromatid migrates to
opposite sides of the cell through the activity of MAPs and motor proteins. The balance of physical
forces established across the chromatid pairs by microtubules ensures a 1:1 segregation of each
chromosome.7
In order to effectively segregate the sister chromatids at anaphase, tubulin subunits must be
able to add to and dissociate from the microtubule. GTP-tubulin is added with a polarity to the
microtubule filament. The faster growing end to which GTP-tubulin adds preferentially is called
the “plus end”; the slower growing end is called the “minus end.” This is called microtubule
treadmilling, and is observed in the mitotic spindle. GTP bound to ß-tubulin is hydrolyzed soon
after it adds to the filament to form guanosine diphosphate (GDP)-tubulin, which has a much larger
dissociation rate constant than its triphosphate form.7
Paclitaxel and docetaxel interfere with the activity of microtubules by binding to the
βtubulin subunit. 9,10,11 When bound, these cytotoxic agents enhance the rate at which β-tubulin
polymerizes, stabilizing the resultant microtubules and thereby inhibiting their depolymerization.
In the presence of these abnormally stable microtubules, the cell cannot effectively function, and
the activity of the mitotic spindle is greatly hindered. As a result, mitotic arrest is induced between
the prophase and anaphase stages of the cell cycle, eventually leading to apoptosis of the cancerous
cells (Figure 1-3).6,8 Paclitaxel also promotes the rate at which tubulin nucleates and polymerizes
(Figure 1-4). Naturally, 13 protofilaments assemble to form a microtubule with a diameter of 24
nm. In the presence of paclitaxel, 12 protofilaments assemble to form a microtubule with a diameter
of about 22 nm. This paclitaxel-microtubule complex is very stable, even under depolymerization
conditions of low temperature or in a CaCl2 solution.9,10,11
5
Figure 1-3 (adapted from [8]). Mitotic arrest induced by paclitaxel and docetaxel between prophase and anaphase.
Figure 1-4 (adapted from [11]). Microtubule formation and the mechanism of action of paclitaxel.
§ 1.2.4 Issues with the Use of Paclitaxel and Docetaxel (Supply, Specificity, and
Ineffectiveness against MDR cancers)
Despite their relative effectiveness against certain types of cancer, namely those of the breast,
ovarian, and lungs, the use of both paclitaxel and docetaxel continues to remain problematic in
several ways. To begin, there is an issue when it comes to the supply of naturally occurring
paclitaxel. Paclitaxel is isolated from the bark of the Pacific Yew tree, Taxus brevifolia, a non-
renewable resource, through an extensive, low-yielding process. As a result, the supply of this drug
is limited to the supply of yew trees, the like of which will become steadily depleted over the
years.6 In addition, neither cytotoxic agent is specific when it comes to the recognition of the
cancerous cells for which they are intended to destroy. As a result, systemic toxicity often results,
leading to the adverse side effects mentioned earlier.4 More so, these drugs are fairly ineffective
6
against cancerous cell lines that express the multidrug resistant (MDR) phenotype, such as colon
carcinoma. The principle mechanism behind MDR cancers has been attributed, at least in part, to
the presence of two molecular pumps in tumor cell membranes that actively expel cytotoxic agents
from their interior.4 One pump, P-glycoprotein (Pgp), is responsible for the drug resistance of colon
carcinoma to common cytotoxic agents.12 Pgp is an effective ATP-binding cassette (ABC)
transporter that effluxes hydrophobic anticancer agents such as paclitaxel and docetaxel (Figure 1-
5). The second pump is referred to as a multidrug resistance-associated protein (MDP).4
Figure 1-5 (adapted from [13]). The Pgp pump is an effective ABC transporter that actively expels various
hydrophobic cytotoxic agents from its interior.13
§ 1.2.5 Resolving the Issue of Supply
Due to the limitations of paclitaxel and docetaxel with regards to supply, specificity, and MDR
cancers, it was imperative to develop new semi-synthetic analogs of these popular cytotoxic agents
in order to resolve, in whole or in part, these issues. The first major advance with respect to the
issue of supply came in 1985, when Potier et al. isolated 10-deacetylbaccatin III (10-DAB III)
from the leaves of the European yew, Taxus baccata (Figure 1-6). This diterpenoid is not only
comprised of the complex tetracyclic core of paclitaxel, it also has the appropriate nine
stereocenters. Since the leaves of the European yew are a renewable resource, the isolation of 10-
DAB III pioneered the use of semi-synthetic methods to secure a long term supply of paclitaxel,
docetaxel, and their analogs.6
7
Figure 1-6. 10-deacetylbaccatin III (DAB), extracted from the leaves of the European yew, Taxus baccata, is the
starting compound in the synthesis of both paclitaxel and docetaxel, as well as other cytotoxic analogs of these drugs.
§ 1.2.6 Resolving the Issue of MDR Cancers Through the Use of 2nd-Generation Taxoids
In tackling the issue of MDR cancers, an excellent place to begin is in the synthesis of a variety
of cytotoxic analogs. These analogs are produced through the coupling of a chiral βlactam to 7-
TES-DAB III, and their relative cytotoxicities are determined through structureactivity
relationship (SAR) studies. Using these studies to their advantage, the Ojima group was able to
determine preferential modifications at the C-3’ position of the isoserine side chain and the C-10
position of the baccatan core of paclitaxel, thereby substantially increasing the cytotoxic potency
of the resultant agents several fold. The increased potency of these so called second generation
taxoids allow them to perform better when faced with MDR cancers.14 SB-T1214, depicted in
Figure 1-7 below, is one such unique second generation taxoid.
Figure 1-7. The Ojima group’s second generation taxoid SB-T-1214 is more potent than paclitaxel in the treatment of
certain MDR cell lines, such as 1A9PTX10 and 1A9PTX22. More so, it has exhibited exemplary pre-clinical results
and has thus been chosen for further study using a targeted conjugate system.15
H O
O H O O H
O O A c O
H H O
O
A B C
D 1 3
1 0
1
7
O
O O H
O O A c O
H H O
O
O
O
O H
N H O
O
O
S B - T - 121 4
8
§ 1.3 β-Lactam Synthesis
§ 1.3.1 β-Lactam
In the past, extensive studies were conducted on the synthesis of β-lactam, a 4 atom heterocyclic
amide, in connection with several naturally occurring antibiotics that bore its core structure in their
chemical make-up. Amongst these antibiotic families are the penicillins, cephalosporins,
carbapenems, and monobactams (Figure 1-8). Collectively, they are known as the β-lactam
antibiotics. These agents work by inhibiting bacterial cell wall synthesis, leading to apoptosis of
the bacterium, especially in the case of gram-positive species. Although extensive
research was conducted on its synthesis, limited attention was drawn to the benefits of the βlactam
structure as an intermediate in the synthesis of other compounds until the advent of the βLSM by
the Ojima group.16 The implementation of this method in the field of drug synthesis and design
allows for the effective synthesis of second generation taxoids, such as SB-T-1216.
R H R2
R3 O COOH
OH O
Penicillin Carbapenem
OSO3H
H2N HO O
O Cephalosporin Monobactam
Figure 1-8. The β-lactam antibiotics contain the β-lactam ring at the core of their chemical structure.
Enantiopure β-Lactam can be prepared in good yield via the chiral ester-enolate imine
cyclocondensation and the Staudinger [2+2] ketene-imine cycloaddition, followed by enzymatic
kinetic resolution. The new generation taxoids are subsequently obtained in the ring opening
coupling of this enantiopure β-lactam to a modified baccatan, followed by deprotection.16
N
S
O
H HN
O
O
S
N S
H HN
O
O
N
1
N N H
O S
N
O O H
N
O
9
y
§ 1.3.2 Synthesis of Enantiopure β-lactams via the Chiral Ester Enolate-Imine
Cyclocondensation
§ 1.3.2.1 Whitesell’s Chiral Auxiliary
Traditionally, chiral β-lactam synthesis through the chiral ester enolate-imine
cyclocondensation started with the synthesis of (-)-trans-2-phenylcyclohexanol through a series of
synthetic reactions to yield racemic trans-2-phenylcyclohexanol followed by enzymatic resolution
with pig liver acetone powder (PLAP) (Scheme 1-1). Two problematic features of this route were
both the overall yield (~35%) and the time (1 week) needed for enzymatic resolution.
Scheme 1-1. Synthesis of Whitesell’s chiral auxiliary through enzymatic resolution with PLAP.
Interestingly, these two problems could be overcome by adapting asymmetric Sharpless
dihydroxylation followed by Raney nickel dehydroxylation (Scheme 1-2). In 1994, Sharpless and
co-workers published a procedure by which (-)-trans-2-phenylcyclohexanol could be obtained by
asymmetric synthesis.17 This procedure was later scaled up by Truesdale and coworkers in 2002.18
Ph Ph
SAD
Ra60ne - 7 Ni0 c%kel OH two steps >
99 % ee
Scheme 1-2. Asymmetric synthesis of Whitesell’s chiral auxillary.
Sharpless designed the use of chiral ligands (DHQD2-PHAL or DHQ2-PHAL) (Figure 19)
derived from the natural product qunine to induce selectivity in the dihydroxylation of internal
alkenes (Scheme 1-3).19 First osmium tetroxide, coordinated to the chiral ligand, underwent a [3 +
10
2] cycloaddition to the olefin to give the 5-membered metallacycle. Under basic conditions,
hydrolysis of this metallacycle liberated the diol while reducing the osmate. Regeneration of the
catalyst by potassium ferricyanide or NMO could be used within the same pot to reoxidize the
catalyst, completing the catalytic cycle. Employing DHQD2PHAL chiral ligand, it was found that
the intermediate (+)-(1R,2S)-1-phenylcyclohexane-cis-1,2-diol could be obtained via Sharpless
dihydroxylation in excellent enantioselectivity (99 % ee).
MeO OMe MeO OMe
(DHQD)2-PHAL (AD-mix-β) (DHQ)2-PHAL (AD-mix-α)
Figure 1-9. Ligands utilized by Sharpless for asymmetric dihydroxylation.
Scheme 1-3. Catalytic cycle of Sharpless asymmetric dihydroxylation.
To selectively remove the alcohol at the benzylic position while providing no reactivity at
the secondary alcohol, a concerted same face reductive hydrogenation was employed using Raney
nickel (Scheme 1-4).17 This reaction is believed to proceed via insertion of nickel into the
C-O bond at the benzylic position followed by reductive elimination to afford (-)-trans-
2phenylcyclohexanol with complete retention of stereochemistry. Sharpless has shown that (-
)trans-2-phenylcyclohexanol can be obtained using this method with enantiomeric excess greater
than 99.5 %.
N
O N
Et N N
O
N
N Et
N
O N N N
O
N
N Et Et
11
Ph + Ni2O3
Intermediate
Scheme 1-4. Selective nickel insertion followed by reductive elimination.
§ 1.3.2.2 Chiral Ester Enolate-Imine Cyclocondensation
Since (-)-trans-2-phenylcyclohexanol could be obtained more readily through asymmetric
catalysis rather than enzymatic resolution, there was an impetus to also improve the chiral ester
synthesis. The original strategy was designed to protect the alcohol end of glycolic acid so that
Whitesell’s chiral auxiliary could be selectively coupled to the carboxylic acid end. This ultimately
led to unnecessary protection and deprotection steps and the use of Pd/C in sizeable quantities. In
addition, low yields after coupling the chiral auxiliary resulted in significant losses and reduction
in recovery of the chiral auxiliary after cyclocondensation. In order to improve upon the
cyclocondensation chiral auxiliary strategy, a new approach was adopted using the developed
triisopropylsilyloxyacetyl chloride (Scheme 1-5).
Scheme 1-5. Revised scheme for asymmetric enolate-imine cyclocondensation.
There are two possible mechanistic pathways by which the formation of cisdemethylvinyl-
β-lactam can occur; E-enolate formation followed by a chair like transition state (A) and Z-enolate
O H Ph
H O H
Ph
H O H
H O H
H Ni H O H
R Ni
I n se r ti o n R e d uc ti v e
Eli m i n a ti o n
C l O TIP S O
O O TIP S O Ph
N O
TIP SO
PMP
O H
Ph
N PMP
12
formation followed by a boat like transition state (B), both of which can accommodate the observed
stereochemical outcome (Figure 1-10).20 While the chiral auxiliary resides in an exo position in B,
it is located in an endo position in A. It is therefore reasonable to assume that transition state A
would bring about much better asymmetric induction than B. Furthermore, it was determined that
E-enolate was kinetically more favorable by 2.5 kcal/mol than B through MM2 calculations using
a MACROMODEL program. Therefore, the formation of E-enolate is preferred in this case.20
Figure 1-10. E-enolate formation and Z-enolate formation with their respective transition states.
Figure 1-11 depicts the chiral ester enolate-imine cyclocondensation mechanism. The chiral
auxiliary, (-)-trans-2-phenyl-cyclohexyl, directs the approach of the trans-imine, N-
(4methoxyphenyl)-3-methyl-2-butenaldimine, from the si-face of the E-enolate (i.e. the least
hindered face), producing the N-lithiated β-amino ester intermediate. Cyclization of this
intermediate releases the chiral alcohol, subsequently producing the desired cis β-Lactam.20
Figure 1-11. The mechanism of chiral ester enolate-imine cyclocondensation.
13
§ 1.3.3 Synthesis of Chiral β-lactams via Staudinger [2+2] Ketene-Imine Cycloaddition
Followed by Enzymatic Kinetic Resolution
The Staudinger [2+2] ketene-imine cycloaddition requires the synthesis of a ketene component
and a trans imine component. The ketene, generated by the reaction of an acyl chloride with an
amine, undergoes a [2+2] cycloaddition with the imine component. The nature of the substituents
residing on the ketene and imine components plays a critical role in determining the relative
stereochemistry of the Staudinger reaction. In the transition state of the conrotatory
electrocyclization, electron donating groups at the terminal carbon atoms favor the outward
position, whereas electron withdrawing groups favor the inward position.21,22 cis βlactam
formation is based on the torquoselectivity of ring closure, in which an electron donating group
residing on the ketene preferentially adopts the outward configuration. Calculations using RHF/6-
31G* have determined that the barrier for conrotatory closure in this manner is 8-12 kcal/mol
lower.23 The outward configuration enables the imine to attack from the least hindered side of the
ketene (i.e. the rear of the R1 group), resulting in the lower energy conrotatory transition structures
and favoring the formation of cis β-lactam (Scheme 1-6).21,24
R2
R3 exo 25
Scheme 1-6. Mechanism of the Staudinger Reaction towards cis β-lactam synthesis.
The Staudinger [2+2] ketene-imine cycloaddition generates a racemic mixture of chiral βlactams.
The two enantiomers are resolved via enzymatic kinetic resolution to afford the desired
enantiopure β-lactam.
C O
R 1 H
N R 3
H R 2 - O N +
R 3
R 2 H
R 1 H N
O R 1
H R 3 H
R 2 N
O
R 1
14
§ 1.4 SB-T-1216 Synthesis
§ 1.4.1 SB-T-1216
SB-T-1216 is a potent second generation taxoid that is more effective than paclitaxel, especially
against breast cancer cell lines expressing MDR phenotypes. Like its parent taxoid, SB-T-1216 is
a microtubule stabilizing agent, generating microtubule bundles in interphase cells.
Due to its increased cytotoxic potency, SB-T-1216 induces microtubule bundle formation
(Figure 1-12) and cell death (Figure 1-13) at lower concentrations than paclitaxel.26
Figure 1-12. Effect of paclitaxel and SB-T-1216 on the formation of interphase microtubule bundles after a 24 h
incubation period in the drug sensitive human breast cancer cell line MDA-MB-435 and the drug resistant human
breast cancer cell line NCI/ADR-RES. Control cells were incubated without taxoid. Microtubules stained with Cy3- conjugated anti-tubulin antibody (red). Cell nuclei stained with DAPI (blue).26
15
Figure 1-13. Effect of SB-T-1216 on the growth and survival of MDA-MB-435 and NCI/ADR-RES cells after a 96 h
incubation period. Control cells (C) were incubated without SB-T-1216. The cells were seeded at 10 x 103 cells/100
μl of medium in the well. The dotted line represents the number of cells of the inoculum. Each point represents the
mean of 8 separate cultures ± SEM.26
Like paclitaxel, SB-T-1216 is also an activator of caspase, a protease that plays an essential role
in programmed cell death.26 The increased potency of SB-T-1216 allows it to induce cell death at
lower concentrations than paclitaxel, especially in the case of drug-resistant cell lines. While the
IC50 (concentration of taxoid resulting in 50% of living cells in comparison with the control) of
SB-T-1216 in the drug-sensitive human breast cancer cell line MDA-MB435 is 0.6 nM, versus 1
nM for paclitaxel, its IC50 in the drug-resistant human breast cancer cell line NCI/ADR-RES is 1.8
nM, versus 300 nM for paclitaxel.26, 27
Due to its impressive cytotoxic efficacy and its effective range against several lines of MDR
cancer, SB-T-1216 is often employed in the synthesis of tumor targeting conjugates for drug
delivery systems.4 These tumor targeting molecules (TTMs) allow for the specific delivery and
uptake of the cytotoxic agent by the intended cancerous cells, largely reducing the incidence of
systemic toxicity and its resultant adverse side effects. Effective and versatile conjugates include
the polyunsaturated fatty acids (PUFAs), such as the docosahexaenoic acid (DHA)-SBT-1216
conjugate.27 The general mechanism by which these tumor targeting conjugates enter and destroy
a cell is depicted in Figure 1-14.
16
Figure 1-14. General receptor mediated endocytosis of a tumor targeting conjugate. Binding of the tumor targeting
recognition moiety to a receptor element on the cancerous cell’s membrane allows for receptor mediated endoctytosis.
Cleavage of the linker within the cell releases the active cytotoxic agent, which subsequently promotes cell death.4
Implementation of the β-LSM has proven to be effective in the synthesis of second generation
taxoids. The β-LSM utilizes the Ojima-Holton protocol to couple the desired chiral βlactam with
high enantioselectivity to a functional baccatan.16 In the synthesis of paclitaxel, 7-
TES-baccatin is coupled to a chiral β-lactam containing a phenyl group at the nitrogen atom of the
ring (Scheme 1-7). In the case of SB-T-1216, the β-lactam used is 1-(tert-butoxycarbonyl)-
3triisopropylsiloxy-4-(2-methylpropen-2-yl)azetidin-2-one, while the employed baccatan is
7TES-10-N,N’-dimethylcarbamoyl-DAB III.
R = t-BuO R1 = Ac Paclitaxel: R = Ph, R1 = Ac R1 = H Docetaxel: R = t-BuO, R1 = H
Scheme 1-7. Ojima-Holton coupling protocol.
17
§ 1.5 Results and Discussion
§ 1.5.1 Synthesis of Whitesell’s Chiral Auxiliary
Synthesis of Whitesell’s chiral auxiliary (WCA) began with the application of Sharpless’s
asymmetric dihydroxylation to 1-phenylcyclohexene to produce (+)-(1R,2R)-
1phenylcyclohexane-cis-1,2-diol 1-I (Scheme 1-8).19 The use of Sharpless’s methodology has been
shown to confer excellent enantiopurity to the desired chiral ester.
PhK3KF2eO(CsNO)46- 2(3H.20O e q(0.).,6 M meoSOl%2)NH, (DH2 (Q1.D0) e2PHAq.), KL2 C(2O.43 m
(3o.0l% e)q.) Ph
OH t-BuOH, H2O (2:3), 0 oC - r.t., 48 h OH
1-I Scheme 1-8. Sharpless asymmetric dihydroxylation of 1-phenylcyclohexene to produce (+)-(1R,2R)-
1phenylcyclohexane-1,2-diol.
After obtaining highly enantiopure cis-diol 1-I, reductive benzylic dehydroxylation was
performed using excess Raney nickel in ethanol to yield highly enantiopure WCA 1-II after
recrystalization (Scheme 1-9).17
PhPh OH Raney Nickel (excess)
OH ethanol, reflux, 5 h OH
1-I 38% over 2 steps> 99% ee 1-II
Scheme 1-9. Preparation of WCA using reductive benzylic dehydroxylation in the presence of Raney Nickel.
Low yield can be attributed to a failure to quantify the remainder of WCA collected from
subsequent recrystalization steps.
§ 1.5.2 Synthesis of β-lactam via Chiral Ester-Enolate Imine Cyclocondensation
18
After obtaining a suitable quantity of 1-II, synthesis of the chiral ester was performed
(Scheme 1-10). The first step in this process involved the silyl-protection of methyl glycolate
using triisopropylsilyl chloride (TIPSCl) in the presence of imidazole and dimethylformamide
(DMF) (Corey protocol) to yield 2-I. Because the silylation reaction is exothermic, the solution
of methyl glycolate and DMF was cooled to 0 °C before the addition of 3 eq. imidazole and the
drop-wise addition of TIPSCl. The solution was then allowed to stir from 0 °C to room
temperature overnight. After obtaining a sufficient quantity of 2-I, hydrolysis of the methyl ester
with aqueous lithium hydride in tetrahydrofuran (THF) afforded selective methyl ester cleavage
without interfering with the silyl-ether TIPS substituent, yielding the free carboxylic acid 2-II.
Upon formation of the free carboxylic acid, treatment with oxalyl chloride in the presence of a
catalytic amount of DMF produced the acyl chloride 2-III.
O DMFimTIPid, aS0z CooCll e( 1t (o.31 r.. 0et q.e,
L(1iO:1H)(-.qo)./)n TIPSO
O OMe
HH22OO: THF(1.5 )e, r.q.t)., o/n
HO OMe
2-I
TIPSO O OH
oxalCylH c2hlClo2r, r.i(dcate. ,t( )o1./3n eq.) TIPSO-
III O Cl DMF 2-II 2
Scheme 1-10. Synthesis of triisopropylsilyloxyacetyl chloride.
Coupling of WCA to the acyl chloride in the presence of pyridine and a catalytic amount
of 4-dimethylaminopyridine (DMAP) at room temperature afforded the desired chiral ester 2-IV
after purification by column chromatography. The HCl generated during the course of this reaction
was trapped by the pyridine salt (Scheme 1-11).
(2-III) (1.15 eq.) DMAP
Ph ridine(
11.1.5 e eq.). Ph
OH CH Cl , r.t. o/n OTIPS
11 %
p y ( q )
2 2 , O
O
19
1-II 2-IV
Scheme 1-11. Coupling of WCA to the TIPS protected acyl chloride in the presence of DMAP and pyridine.
Since the subsequent cyclocondensation reaction is sensitive, the chiral ester must be
extremely pure. Low yield can be attributed to the loss of material during purification, as two
sequential columns were utilized in order to afford the pure chiral ester. The reported yield is based
on the purest of fractions collected from the second column. In actuality though, a greater amount
product was collected, although relatively less pure.
In order to derive the appropriate chiral β-lactam via the chiral ester-enolate imine
cyclocondensation, the appropriate trans-PMP imine component of the reaction was prepared by
aldehyde-amine condensation via dehydration with anhydrous magnesium sulfate. Previous
studies by Ojima et al. have reported that p-anisidine preferentially reacts with 3-methyl-2butenal
to form the trans-imine N-(4-methoxyphenyl)-3-methyl-2-butenaldimine 2-V (Scheme 112).28 Due
to its instability, it was important to keep the imine in a cool, dark, and dry environment to prevent
its hydrolysis.
NH2 2.0 e .
O
Scheme 1-12. Synthesis of a trans imine via aldehyde-amine condensation.
The resultant compound 2-V then underwent a cyclocondensation reaction with the TIPS
protected chiral ester 2-IV, forming (+)-cis-(2-methylprop-1-enyl)-β-lactam 2-VI. (Scheme
113).20
Ph
Ph OTIPS
OOH
2-IV O
2-VI 1-II
O
M g SO 4 ( q ) 3 m - e th y l - 2 - b u t e n a l ( 1 . 1 e q . )
C H 2 C l 2 , r. t . , 3 h r
2 - V
N
L DA ( 1 . 3 e q . )
THF , - 7 8 o C , 3 h
L iHMD S ( 1 . 0 e q . ) , - 4 0 o C , 0 . 5 h
+ N O
TIP SO ( 2 - V ) ( 1 . 3 e q . )
O
20
Scheme 1-13. The chiral ester enolate-imine cyclocondensation is carried out using the TIPS protected chiral ester
and the trans imine to produce (+)-cis-(2-methylprop-1-enyl)-β-lactam.
The resultant cis β-lactam was then subjected to PMP deprotection by cerium ammonium nitrate
in acetonitrile/water at -10°C to yield the desired TIPS protected cyclic amide (Scheme 113).16
PMP deprotection is achieved in a three step mechanistic process, two of which involves a single
electron transfer (SET). The first step proceeds through a single electron transfer as Ce (IV)
removes an electron from the para position to produce a radical/cation intermediate. This
intermediate is susceptible to nucleophilic attack by a water molecule, leading to the formation of
methanol. A second electron transfer takes place as another equivalent of Ce (IV) removes an
electron at the para position to generate a cationic species. The positive charge is subsequently
neutralized by hydrolysis at the ipse position of the phenyl ring, leading to cleavage of the C-N
bond to produce a quinone molecule and liberating the free amine 2-VII.16,29 2-VII was then treated
with di-tert-butyl dicarbonate (Boc) under basic conditions to afford the desired N-Boc protected,
enantiopure β-lactam 2-VIII (Scheme 1-14).16
TIPSO TIPSO
OO
2-VI O 2-VII
20% over 2 steps
2-VIII
Scheme 1-14. Boc protection of the free amide in the presence of Boc anhydride and a catalytic amount of DMAP.
Low yield can be attributed, at least in part, to a lack of ideal reaction conditions during the CAN
PMP deprotection step. In lieu of dry ice, the reaction was cooled to 0 °C in an ethanol/salt ice
bath. As the ice melted, the temperature rose to ~-4 °C, where it stayed for the greater part of the
reaction, reaching 0 °C near the reaction’s completion. After purification by column
NH ce r i c a mm o n i u m n it r a t e ( 4 . 0 e q . )
( 1 : 1 )( M e C N : H 2 O ) [ 0 . 0 2 M ] , - 1 0 o C , 1 . 5 h
N
B oc 2 O ( 1 . 25 e q . )
DMAP ( 0 . 3 e q . ) TEA ( 2 . 0 e q . )
C H 2 C l 2 , r. t . , o / n N
TIP SO
O O
O
21
chromatography, the resultant compound 2-VII was still relatively impure. These impurities could
have played a role in affecting the subsequent BOC reaction.
Synthesis of highly enantiopure β-lactam via chiral ester enolate-imine cyclocondensation is a
versatile and practical route towards the synthesis of taxoids.
§ 1.5.3 Synthesis of β-lactam via Staudinger [2+2] Ketene-Imine Cycloaddition Followed by
Enzymatic Resolution
Synthesis of the trans-imine component was carried out by the same method as depicted in
Scheme 1-12. Subsequent reaction of this imine with acetoxyacetal chloride and triethylamine
(TEA) yields the β-lactam ring via a Staudinger [2+2] ketene-imine cycloaddition (Scheme 115).
The ketene is first generated by the reaction of acetoxyacetal chloride with triethylamine, followed
by the nucleophilic addition of the imine nitrogen atom to the central carbon of the ketene.
Alternatively, the ketene can act as the nucleophile and add to the electrophilic center of the imine.
The result of either approach generates a zwitterionic intermediate, which subsequently undergoes
electrocyclic conrotatory ring closure to afford the β-lactam ring (±) 3I.21,22 Stereoselectivity is
derived from the stereoarrangement of groups generated during the transition state. The reaction
is most favorable when carried out under very low temperature conditions, as this both increases
the yield and decreases the formation of substantial byproducts.
O O
Na
SO 2 e H2N .1 eq.) CH2Cl2, r.t., 3 h 2-V TEACH (12C.6l 2eq.) O
(1 -78 °C - r.t., overnight
62% over 2 steps (+/-) 3-I
Scheme 1-15. Staudinger [2+2] ketene-imine cycloaddition.
O
H
O
+ 2 4 ( q )
O O
C l
( 1 . 2 e q ) N
O N
O
O
22
The racemic (+/-) 3-I was generated in good yield (62% over 2 steps) after purification by column
chromatography. PS Amano Lipase, an enzyme derived from the bacterial species Burkholderia
cepacia, preferentially hydrolyzes the (-) enantiomer while remaining uncreative towards the (+)
enantiomer. This selective hydrolysis occurs under physiological pH conditions to afford the
hydrolyzed (-) alcohol, (-) 3-II, and the desired (+) enantiomer of 3-I (Scheme 116).29 The extent
of the reaction was monitored by 1H NMR until 50% conversion and by chiral, normal phase HPLC
to ensure high enantioselectivity.
O O
HO
+ O
(+/-) 3-I 85% (+) 3-I O
(-) 3-II O
Scheme 1-16. Enzymatic Resolution.
The desired enantiomer (+) 3-I was produced in good yield (85%) and with excellent enantiomeric
excess (> 99% ee). The acetate group of the enantiopure β-lactam was subjected to hydrolysis in
the presence of potassium hydroxide to generate (+) 3-II. Subsequent TIPS protection of this
hydroxyl group was carried out with the use of TIPSCl in the presence of TEA and a catalytic
amount of DMAP to afford (+) 3-III (Scheme 1-17).
O
(+) 3-I
O (+) 3-II (+) 3-III
O
Scheme 1-17. Acetate hydrolysis and TIPS protection of cis β-lactam.
Hydrolysis of the acetate group and subsequent TIPSCl protection affords the desired chiral β-
lactam in good yield (85%) after purification by column chromatography. The PMP deprotection
and Boc protection undergo an approach similar to the method described in section
N
O
O
O
2 0 % P S A m a n o L i p ase PB S , p H 7 . 5
1 0 % C H 3 C N i n H 2 O
4 5 ° C , 1 0 d
N O
O
N
23
1.5.2. Synthesis of β-lactam via Staudinger [2+2] ketene-imine cycloaddition followed by
enzymatic resolution is an efficient method as a precursor towards taxoid synthesis.
§ 1.5.4 Synthesis of SB-T-1216
Synthesis of SB-T-1216 began with functionalization of 10-DAB III (Scheme 1-18). In
order to selectively acylate the C-10 and C-13 positions of 10-DAB III, the hydroxyl group on C7
must first be protected using chlorotriethylsilane (TESCl), as the most acidic proton resides there.
Protection of the hydroxyl groups residing at the C-10 and C-13 positions is avoided by limiting
the length of the reaction and the equivalents of TESCl used. The hydroxyl group residing at the
C-1 position is too sterically hindered by the benzyl group at C-2, and hence does not compete
with the C-7 alcohol for acylation.14 Protection of the hydroxyl group at C-7 was carried out at 0°
C with the use of excess TESCl and imidazole (Corey protocol) to yield protected DAB III 4-I.
Imidazole functions to deprotonate the alcoholic proton at the C-7 position, resulting in an SN2
attack of the resultant nucleophilic alkoxide towards TESCl and the displacement of the chloride
ion.14
Having selectively protected the C-7 position of 10-DAB III, the hydroxyl residing at the
C-10 position is now the most reactive group of the baccatan core. The mono-TES baccatan was
treated with LiHMDS at -40 °C, and the resultant lithium-10-alkoxysalt reacted with
N,N’dimethylcarbamoyl chloride via an addition-elimination pathway to afford 7-TES-10-
N,N’dimethylcarbamoyl-DAB III 4-II.14
10-DAB III 4-I
24
O
O
LiHMDS (1.0 eq) THF, -40
oC
89%
4-II 14
Scheme 1-18. Synthesis of 7-TES-10-N,N’-dimethylcarbamoyl-DAB III.
Low yield of 4-I (80%) can be attributed to the formation of di- and tri-TES byproducts, which
were removed upon purification of 4-1. This substantial byproduct formation was likely due to the
addition of a little over 3.0 eq. of TESCl to the reaction. 4-II was produced in good yield (89%)
upon purification by column chromatography.
Upon generating compounds 2-VIII and 4-II, SB-T-1216 was synthesized via the
OjimaHolton coupling protocol and subsequent silyl deprotection (Scheme 1-19)14. First, 1.2
equivalents of LiHMDS, followed by 1.2 equivalents of (+) 2-III, was added to 4-II in THF at -
40 °C. The resultant lithium-13-alkoxysalt attacks and opens up the β-lactam ring, generating 4III.
The final step in the synthesis of SB-T-1216 involves the deprotection of the C-2’ and C-7
protecting groups with HF/pyridine. The exothermic reaction was carried out at °C and allowed to
proceed to room temperature overnight. Purification via silica gel column chromatography
followed by re-crystallization from ether anhydrous afforded purified SB-T-1216 (4-IV).
H O
O O TE S
O O A c O
H H O
O
O N
C l ( 1 . 0 e q ) N
25
Scheme 1-19: Synthesis of SB-T-1216 through Ojima-Holton coupling of 7-TES-10-N,N’-dimethylcarbamoyl-DAB III with 1-(tert-Butoxycarbonyl)-3-triisopropylsiloxy-4-(2-methylpropen-2-yl)azetidin-2-one, followed by
deprotection.14
Low yield of 4-III (64%) can be attributed, in part, to a lack of ideal reaction conditions. The THF
was relatively wet, as it was obtained from a bottle of previously distilled THF that had been stored
at room temperature for several weeks prior to its use. The LiHMDS was also of poor quality,
being obtained from a desiccator rather than its ideal place of storage in the freezer. More so, it
was a dark orange color rather than its characteristic pale yellow color. 97 mg of starting material
4-II was recovered after purification of 4-III by column chromatography. 4-IV was obtained in
excellent yield (92%) upon purification by column chromatography and recrystalization from
ether.
§ 1.6 Experimental
General information
All chemicals were obtained from Sigma-Aldrich, Fisher Scientific or VWR International, and
used as is unless otherwise noted. All reactions were carried out under nitrogen in oven dried
glassware using standard Schlenk techniques unless otherwise noted. Reactions were monitored
26
by thin layer chromatography (TLC) using E. Merck 60F254 precoated silica gel plates and
alumina plate depending on the compounds. Dry solvents were degassed under nitrogen and were
dried using the PURESOLV system (Inovatative Technologies, Newport, MA). Tetrahydrofuran
was freshly distilled from sodium metal and benzophenone. Dichloromethane was also distilled
immediately prior to use under nitrogen from calcium hydride. Toluene was also distilled
immediately prior to use under nitrogen from calcium hydride. Yields refer to chromatographically
and spectroscopically pure compounds. Flash chromatography was performed with the indicated
solvents using Fisher silica gel (particle size 170-400 Mesh).1H, 13C and 9F data were obtained
using either 300 MHz Varian Gemni 2300 (75 MHz 13C, 121 MHz
19F) spectrometer, the 400 MHz Varian INOVA 400 (100 MHz 13C) spectrometer or the 500 MHz
Varian INOVA 500 (125 MHz 13C) in CDCl3 as solvent unless otherwise stated. Chemical shifts
(δ) are reported in ppm and standardized with solvent as internal standard based on literature
reported values. Melting points were measured on Thomas Hoover Capillary melting point
apparatus and are uncorrected. Optical rotations were measured on Perkin-Elmer Model 241
polarimeter.
Experimental Procedure
(+)-(1R,2S)-1-Phenylcyclohexane-1,2-diol [1-I]:
To a 500 mL round-bottom flask was added 36.9 g (133 mmol) potassium ferricyanide, 15.5 g (133
mmol) potassium carbonate, and 4.1 g (44 mmol) methanesulfonamide. Then 50 mL of tertbutanol
in 70 mL of distilled water was added. The contents of the solution were allowed to homogenize
through vigorous stirring with a magnetic stir bar, and the reaction was cooled to 0 °C in an ice
bath. To this stirring solution was added 97.0 mg (0.26 mmol) of potassium osmate dehydrate and
0.71 g (1.06 mmol) of (DHQD)2-PHAL ligand. After stirring for an additional 20 minutes, 7 ml
(44 mmol) of 1-phenylcyclohexene was added to the solution dropwise. This solution was allowed
to stir from 0 °C to room temperature over the course of 48 hours. The reaction mixture visibly
changed from a dark red-orange color to a light yellow color during this period as the potassium
ferricyanide was reduced by the catalyst. After completion, 25 ml of ethyl acetate was added to the
solution, and the solution was allowed to stir for an additional 15 minutes. The entire solution was
then filtered through a bed of celite to remove solid potassium ferrocyanide. The organic layer was
27
extracted with ethyl acetate, washed three times with water, dried over anhydrous MgSO4, filtered,
and concentrated in vacuo to afford 1-I as a slightly yellow-tinted white solid.
(-)-trans-2-Phenylcyclohexanol (WCA) [1-II]:
To a 1000 mL round-bottom flask containing 20 g (104 mmol) of 1-I was added 250 mL of ethanol.
This solution was allowed to stir with the use of a mechanical stirring rod until the solid was
dissolved. The reaction flask was purged with N2. To this solution was added 300 mL of Raney®-
Nickel 2800 catalyst. The reaction flask was then equipped with a reflux condenser and heated to
100 °C for 5 hours. The reaction was monitored via TLC (3:1 hexanes/ethyl acetate, stain- PMA)
with the diol appearing at an Rf of 0.4 and the dehydroxylated product appearing at an Rf of 0.6
After completion, the reaction was cooled to room temperature and then filtered through a bed of
celite while taking care not to dry the solution, as the pyrogenic Raney Nickel would have ignited
under arid conditions. The resulting black Ni solid was washed with copious amounts of ethanol
and then diluted with water before proper disposal. Solvent was removed from the collected
fraction via rotary evaporation. The organic layer was extracted with ethyl acetate, washed two
times with water, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford a
white solid. Due to the paste-like consistency of the product, it was determined that there was a
remnant of water in the flask. Thus, the compound was extracted with CH2Cl2, washed two times
with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford a light
yellow oil, which spontaneously solidified to form white crystals after a brief cooling period. This
solid was then dissolved in a small amount of warm pentane and allowed to re-crystallize at 0 °C
for 1 hour to afford 1-II (6.97 g, 38% over 2 steps). The solid crystals were then washed several
times with chilled pentanes and dried using paper filtration. The collected white crystals were re-
dissolved in a small amount of warm pentane and allowed to re-crystallize at 0 °C for another hour.
The resultant white crystals were long and thin. The purity of the desired product was ascertained
by normal parameterization HPLC with > 99% ee of the (1R,2S) enantiomer (ChiralCel-OD 1;
flow rate: 0.4 ml/min; injection volume: 10 ml; 98% hexanes/2% ethyl acetate). The mother liquor
collected from the paper filtration step was subjected to rotary evaporation and the recrystalization
process re-performed so as to salvage additional product. Further enantiomeric enrichment was
performed on the second recrystalization. 1H NMR (500 MHz, CDCl3) δ 1.32-1.56 (m, 6H), 1.85-
28
1.88 (m, 2H), 2.12-2.14 (m, 1H), 2.41-2.46 (m, 1H), 3.68 (s, 1H), 7.23-7.27 (m, 3H), 7.32-7.35 (t,
2H, J = 7.5 Hz). All data were found to be in agreement with literature values.
Triisopropylsilyl-oxymethylglycolate [2-I]:
To a 250 mL round-bottom flask under continual N2 purging was added 5.004 g (55.55 mmol)
methyl glycolate and 11.368 g (166.7 mmol) imidazole. Then 18.0 mL of dry DMF (56 mmol) was
added and the solution was allowed to stir until homogenous in a 0 °C ice bath. To this solution
was added 13.1 mL (61.11 mmol) of TIPSCl dropwise (about 12.5 ml of the TIPSCl was of poor
quality, being a faint yellow color, the remaining 0.6 ml of TIPSCl was its characteristic clear
color). As the reaction proceeded, the solution became a dilute, milky white, evidence of the
imidazole salt precipitation. The reaction was allowed to proceed for 22 hours, going from 0 °C to
room temperature overnight under continual stirring. Upon completion, the reaction was quenched
with saturated ammonium chloride (15 mL). The organic layer was extracted with ethyl acetate,
washed four times with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to
afford 2-I, a slightly yellow oil. This oil was pure enough by 1H NMR for the next synthetic step
without further purification. 1H NMR (300 MHz, CDCl3) δ 1.03-1.15 (m, 21H), 3.70 (s, 3H), 4.29
(s, 2H). All data were found to be in agreement with literature values.
Triisopropylsilyl-oxyacetic acid [2-II]:
To a 250 mL round-bottom flask under the continual purging of N2 was added 14.519 g (58.92
mmol) of 2-I and 90 mL of THF. The solution was allowed to stir in a 0 °C ice bath, followed by
the dropwise addition of 3.652 g (88.39 mmol) of LiOH-H2O dissolved in 90 ml of distilled water.
The reaction was allowed to proceed from 0 °C to room temperature over the course of 72 hours.
During this time, the solution changed from a dark yellow color to a clear color. The pH of the
solution was lowered to an acidic pH of 3.0 by the slow addition of a 1N solution of aqueous HCl.
During the course of this addition, the solution became a cloudy white as Li+Cl- salt precipitated.
The organic layer was extracted with CH2Cl2, washed three times with brine, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo to afford 4-II, a light yellow oil. This oil was pure
enough by 1H NMR for the next synthetic step without further purification. 1H NMR (300 MHz,
CDCl3) δ 1.06-1.15 (m, 21H), 4.29 (s, 2H). All data were found to be in agreement with literature
values.
29
Triisopropylsilyl-oxyacetyl chloride [2-III]:
To a 500 mL round-bottom flask under the continual purging of N2 was added 12.083 g (51.99
mmol) of 2-II, followed by the addition of 100 mL of dry dichloromethane (DCM). The solution
was allowed to stir in a 0 °C ice bath. To this solution was added 6.0 mL (67 mmol) oxalyl chloride
dropwise, and the resulting solution was allowed to stir until homogenous. Then, 0.1 ml of DMF
(30 drops) was added, and the resulting solution was allowed to stir from 0 °C to room temperature
overnight. After completion, the organic layer was concentrated in vacuo to afford 2-III. The
resultant compound was a light yellow liquid with a precipitate of dark yellow-orange salt crystals
at the bottom of the flask. The yellow liquid was pipetted off from these crystals and placed in a
separate vial. This liquid was used in the subsequent step without further purification. 13C NMR
(300 MHz, CDCl3) δ: 13.93 (CH3), 17.98 (CH), 70.44 (CH2), 77.34 (CO). All data were found to
be in agreement with literature values.
(1R,2S)-(-)-2-Phenylcyclohexyl-triisopropylsilyl-oxyacetate [2-IV]:
To a 250 mL round-bottom flask under the continual purging of N2 was added 4.95 g (28.1 mmol)
of 1-II, followed by the addition of 70 ml CH2Cl2 and 3.777 g (30.88 mmol) of DMAP. The
solution was allowed to stir until homogenous, then, 3.5 ml (42 mmol) of pyridine was added,
followed by the dropwise addition of 8.751 g (32.29 mmol) of 2-III. As 2-III was added to the
solution, it changed from a clear to a light yellow color. The reaction was allowed to proceed at
room temperature for 72 hours. The reaction was monitored via TLC (solvent- 9:1 hexanes/ethyl
acetate, stain- PMA). After completion, the reaction was quenched with 10 mL of saturated sodium
bicarbonate. The organic layer was extracted with DCM, washed three times with brine, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo to afford a dark brown oil. Purification of
this crude oil was performed by column chromatography on silica gel with the incremental increase
of ethyl acetate (1.0% - 2.5% ethyl acetate/hexanes). The purification was monitored via TLC
(solvent- 9:1 hexanes/ethyl acetate, stain- PMA). A second column was performed for further
purification after assessing the quality of the first collected fraction of product. The column was
run on silica gel with an incremental increase of ethyl acetate (0.5% - 2.5% ethyl acetate/hexanes).
The purification was monitored via TLC (solvent- 9:1 hexanes/ethyl acetate, stain- PMA). It was
determined that fractions 17-23 contained the desired product. These fractions were collected and
30
concentrated in vacuo to afford 2-IV (1.188 g, 11%) a relatively clear liquid with a faint tint of
light yellow. Based on NMR data, it was determined that this product was pure enough for use in
the subsequent cyclocondensation reaction. 1H NMR (600 MHz, CDCl3) δ 0.96-1.06 (m, 21H),
1.29-1.58 (m, 6H), 1.77-1.79 (d, 1H, J = 6.9 Hz), 1.84-1.86 (d, 1H, J = 1.2 Hz), 1.92-1.94 (d, 1H,
J = 6.6 Hz), 2.12-2.15 (d, 1H, J
= 7.8 Hz), 3.90-3.92 (d, 1H, J = 8.1 Hz), 4.06-4.08 (d, 1H, J = 8.4 Hz), 7.15-7.20 (m, 3H), 7.237.26
(m, 2H, J = 9.0 Hz). All data were found to be in agreement with literature values. The fractions
that contained un-reacted WCA were collected and re-crystallized.
N-(4-Methoxyphenyl)-3-methyl-2-butenaldimine [2-V]:
To a 25 ml round-bottom flask under the continual purging of N2 was added 250 mg (2.03 mmol)
of p-ansidine, followed by the addition of 1.221 g of MgSO4. The reaction flask was covered with
aluminum foil, as 3-methyl-2-butenal is sensitive to both heat and light. To this flask was added
5.0 ml of CH2Cl2, followed by the dropwise addition of 0.2 ml of 3-methyl-2-butenal. The reaction
mixture was allowed to stir at room temperature for 3 hours and monitored via TLC (solvent- 9:1
hexanes/ethyl acetate). After completion, the solid MgSO4 was removed by filtration. Solvent was
removed from the collected fraction using a rotary evaporator to yield 2V, a light yellow solution,
which was then immediately used in the subsequent step without further purification. 1H NMR
(300 MHz, CDCl3) δ 1.96 (s, 3H), 2.01 (s, 3H), 3.81 (s, 3H), 6.196.23 (d, 1H, J = 10.8 Hz), 6.87-
6.90 (d, 2H, J = 6.6 Hz), 7.10-7.13 (d, 2H, J = 9.0 Hz), 8.37-8.40 (d, 1H, J = 9.6 Hz). All data were
found to be in agreement with literature values.
(3R,4S)-1-(4-Methoxyphenyl)-3-triisopropylsilyloxy-4-(2-methylprop-1-enyl)-azetidin-2-one
[2-VI]:
To a 15 mL round-bottom flask under the continual purging of N2 was added 1.0 ml of THF and
0.50 mL (0.83 mmol) of lithium diisopropylamide (LDA). The solution was then cooled down to
-78 °C in a dry ice/acetone bath and allowed to stir. To this flask was added 250 mg (0.641 mmol)
of chiral ester dissolved in 1.0 ml of THF over the course of 22 minutes. The flask which contained
the chiral ester was rinsed with an additional 1.0 ml of THF and this was added to the solution
dropwise over the course of 4 minutes. The reaction was allowed to proceed for 1.5 hours to form
the enolate. To this solution was added 159 mg (0.833 mmol) of 2-V dissolved in 1.0 ml of THF
31
dropwise over the course of 25 minutes. By this time, the imine had become a dark orange-brown.
The flask which contained the imine was rinsed with an additional 1.0 ml of THF and this was
added to the solution dropwise over the course of 5 minutes. The reaction was allowed to proceed
for 1 hour and monitored via TLC (solvent- 9:1 hexanes/ethyl acetate, stain- vanillin). The reaction
was cooled to ~-40 °C, and 250 μl of lithium bis(trimethylsilyl)amide (LiHMDS) was added to the
solution. The reaction was allowed to proceed for an additional 30 minutes and monitored via TLC
(solvent- 9:1 hexanes/ethyl acetate, stain- vanillin). At completion, the reaction was quenched with
2 mL of saturated ammonium chloride. The organic layer was extracted with ethyl acetate, washed
three times with saturated ammonium chloride, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo to afford 2-VI (182 mg), a crude brown oil with evidence of crystal
formation. This crude product was purified by recrystalization in warm pentanes, and placed at 0
°C overnight. 1H NMR (300 MHz, CDCl3) δ 1.10-1.15 (m, 21H), 1.80 (s, 3H), 1.84 (s, 3H), 3.77
(s, 3H), 4.78-4.83 (dd, 1H, J = 4.8, 5.1 Hz), 5.05-5.06 (d, 1H, J = 5.1 Hz), 5.31-5.34 (d, 1H, J =
9.9 Hz), 7.27-7.30 (m, 4H). All data were found to be in agreement with literature values.
3-Triisopropylsilyloxy-4-(2-methylpropen-2-yl)azetidin-2-one [2-VII]:
To a 250 ml 2-necked round-bottom flask was added 932 mg of 2-IV (2.31 mmol) dissolved in 40
mL of acetonitrile. The solution was cooled to -10 °C in an ethanol/salt ice bath while stirring.
After allowing the solution to cool, 5.070 g (9.24 mmol) of ceric ammonium nitrate (CAN)
dissolved in 40 mL of H2O was added dropwise via an addition funnel (~1 drop/5 seconds over
the course of 30 minutes). The solution became a deep orange color upon addition of CAN. The
reaction was monitored via TLC (solvent- 3:1 hexanes/ethyl acetate; stain- vanillin). The reaction
was allowed to proceed for 3 hours. Upon completion, the organic layer was extracted in ethyl
acetate, washed three times with brine, two times with sodium sulfite, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo to afford a dark brown oil. Purification of this crude
oil was performed by column chromatography on silica gel, with an increasing gradient of ethyl
acetate in hexanes (6% - 20% ethyl acetate/hexanes). Purification was monitored via TLC
(solvent- 3:1 hexanes/ethyl acetate; stain- vanillin). Fractions 58-75 were collected and
concentrated in vacuo to afford 2-VII (273 mg) as light orange crystals. 1H NMR (300 MHz,
CDCl3) δ 1.10-1.15 (m, 21H), 1.76 (s, 3H), 1.83 (s, 3H), 4.99-5.01 (dd, 1H, J = 2.1, 2.4 Hz), 5.21
(m, 1H), 5.38 (m, 1H), 6.86 (s, 1H). All data were found to be in agreement with literature values.
32
1-(tert-Butoxycarbonyl)-3-triisopropylsiloxy-4-(2-methylpropen-2-yl)azetidin-2-one [2VIII]:
To a 50 ml round-bottom flask was added 275 mg of 2-VII (0.925 mmol) and 34 mg of DMAP
(0.28 mmol) dissolved in 5.2 mL of CH2Cl2. The solution was cooled to 0 °C in an ice bath while
stirring before the addition of 0.26 ml (1.8 mmol) of triethylamine (TEA). To this solution was
added 261 mg (1.16 mmol) of di-tert-butyl dicarbonate (Boc) dissolved in 2 ml of CH2Cl2
dropwise; the solution became a brown color after all components were added. The reaction was
allowed to proceed from 0 °C to room temperature overnight. Upon completion, the resulting
solution, now a darker shade of brown, was quenched with saturated ammonium chloride. The
organic layer was extracted with DCM, washed two times with brine, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo to afford a brown oil. Purification of this crude oil was
performed by column chromatography on silica gel, with an increasing gradient of ethyl acetate in
hexanes (0.5% - 2% ethyl acetate/hexanes). The purification was monitored via TLC (solvent- 3:1
hexanes/ethyl acetate; stain- vanillin). Fractions 19-38 were collected and concentrated in vacuo
to afford the desired product 2-VIII (221 mg, 20% over 2 steps) as a relatively clear oil.
(±)-1-(4-Methoxyphenyl)-3-acetoxyl-4-(2-methylprop-1-enyl)azetidin-2-one [3-I]:
To a 250 ml 2-necked round-bottom flask under the continual purging of N2 was added 10 g of 2-
V (53 mmol) dissolved in 100 ml of CH2Cl2. The solution was cooled to -78 ˚C in an acetone/dry
ice bath while stirring vigorously. To this solution was added 12.6 ml of TEA (84.5 mmol)
dropwise, followed by the dropwise addition of 7.3 ml of acetoxy acetal chloride dissolved in 8 ml
of DCM via a mechanical syringe pump over the course of 1-2 hours. The reaction was allowed to
proceed from -78 °C to room temperature overnight, becoming dark brown. Upon completion, the
reaction was quenched with saturated ammonium chloride. The organic layer was extracted in
DCM, washed two times with brine, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo to afford a dark brown oil. Purification of this oil was performed by column chromatography
on silica gel, with an increasing gradient of ethyl acetate in hexanes (10% - 35% ethyl acetate in
hexanes). Purification was monitored via TLC (solvent- 3:1 hexanes/ethyl acetate; stain- vanillin).
Fractions 72-86 were collected and concentrated in vacuo to afford an off-white solid. Further
purification was performed by washing the product with warm hexanes and decanting off the
solvent to afford racemate 3-I
33
(9.40 g, 62% over 2 steps) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.79 (s, 3H), 1.82 (s,
3H), 2.11 (s, 3H), 3.78 (s, 3H), 4.95-4.99 (dd, 1H, J = 3.6, 3.6 Hz), 5.12-5.15 (d, 1H, J = 9.0 Hz),
5.80-5.81 (d, 1H, J = 3.6 Hz), 6.85-6.87 (d, 2H, J = 6.9 Hz), 7.31-7.33 (d, 2H, J = 6.9 Hz). All data
were found to be in agreement with literature values.
Enzymatic Resolution of 3-I
To a 3-necked round-bottom flask under continual N2 purging was added 11.1 g (38.4 mmol) of 3-
I dissolved in 140 ml of 1:1 acetonitrile:H2O, followed by the addition of 1.4 L of a 0.2 M
potassium phosphate buffer. The solution was allowed to stir vigorously and warm to 45 °C in an
oil bath. After reaching this temperature, 2.22 g of PS Amano Lipase was added. The solution was
a light brown/tan color after the addition of all components, and heterogeneous in nature. The
reaction was monitored over the course of 10 days via TLC and 1H NMR until 50% conversion of
the acetate moiety and the hydroxyl moiety had been achieved. Upon completion, the reaction was
filtered through a bed of celite to remove the enzyme. The organic layer was extracted with ethyl
acetate, washed three times with brine, dried over anhydrous MgSO4, filtered, and concentrated in
vacuo to afford a dark brown oil. Purification of this oil was performed by column chromatography
on silica gel, with an increasing gradient of ethyl acetate in hexanes (10% - 26% ethyl
acetate/hexanes). Fractions 10-39 were collected and concentrated in vacuo to afford enantiopure
(+) 3-I (5.02 g, 85%) and the resulting alcohol (-) 3-II. 1H NMR
(300 MHz, CDCl3) δ 1.80 (s, 3H), 1.82 (s, 3H), 2.11 (s, 3H), 3.79 (s, 3H), 4.95-5.0 (dd, 1H, J =
4.5, 5.1 Hz), 5.12-5.15 (d, 1H, J = 10.8 Hz), 5.80-5.81 (d, 1H, J = 3 Hz), 6.84-6.88 (m, 2H), 7.30-
7.33 (m, 2H). All data were found to be in agreement with literature values. After performing a
small scale hydrolysis of (+) 3-I, the enantiomeric excess was ascertained by normal
parameterization HPLC with > 99% ee (ChiralCel-OD 1; flow rate: 0.6 ml/min; injection volume:
10 μl; 85% hexanes/15% isopropanol). (3R,4S)-1-(4-Methoxyphenyl)-3-hydroxy-4-(2-
methylprop-1-enyl)azetidin-2-one [(+) 3-II] To a 500 ml round-bottom flask was added 4.544
g (15.72 mmol) of (+) 3-I dissolved in 300 ml of THF (which was a faintly light brown color rather
than its characteristic clear color). The solution was cooled to 0 °C in an ice bath while stirring. To
this mixture was added 90 ml of 1M KOH (aq) dropwise via an addition funnel. The solution was
a light brown/tan color after the addition of all components. The reaction was allowed to proceed
for 4 hours and monitored via TLC (solvent- 1:1 hexanes/ethyl acetate; stain- vanillin). Upon
34
completion, the reaction was quenched with saturated ammonium chloride, extracted with DCM,
washed two times with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to
afford (+) 3-II (3.62 g, 93%) as a relatively white solid. 1H NMR (300 MHz, CDCl3) δ 1.95 (s,
3H), 2.00 (s, 3H), 3.81 (s, 3H), 5.29 (s, 1H), 6.18-6.23 (d, 1H, J = 12.6 Hz) 6.87-6.91 (m, 2H),
7.09-7.13 (m, 2H), 8.378.40 (d, 1H, J = 9.6 Hz). All data were found to be in agreement with
literature values.
(3R,4S)-1-(4-Methoxyphenyl)-3-triisopropylsilyloxy-4-(2-methylprop-1-enyl)-azetidin-2-one
[(+) 3-III]
To a 250 ml round-bottom flask under continual N2 purging was added 3.62 g (14.7 mmol) of (+)
3-II and 539 mg (4.39 mmol) of DMAP dissolved in 150 ml of CH2Cl2. The solution was cooled
to 0 °C in an ice bath while stirring. After 10 minutes, 4.1 ml (29.29 mmol) of TEA was added
dropwise, followed by the dropwise addition of 4.7 ml (21.97 mmol) of TIPSCl. The solution was
relatively clear, with a tint of yellow. The reaction was allowed to proceed from 0 °C to room
temperature overnight, and monitored via TLC. Upon completion, the reaction was quenched with
saturated ammonium chloride. The organic layer was extracted with DCM, washed three times
with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford a slightly
yellow solid. Purification of this solid was performed by washing in warm hexanes and decanting
off the solvent to afford (+) 3-III (5.02 g, 85%) as a white solid. 1H NMR (300 MHz, CDCl3) δ
1.05-1.15 (m, 21H), 1.80 (s, 3H), 1.84 (s, 3H), 3.77 (s, 3H), 4.78-4.83 (dd, 1H, J = 4.8, 5.1 Hz),
5.05-5.06 (d, 1H, J = 5.1 Hz), 5.31-5.35 (d, 1H, J = 9.9 Hz), 7.27-7.30 (m, 4H). All data were
found to be in agreement with literature values. 7-Triethylsilyl-10-deacetylbaccatin III [4-I]:
To a 50 ml round-bottom flask under continual N2 purging was added 1.0 g (1.8 mmol) of 10DAB
III and 500 mg (7.3 mmol) of imidazole dissolved in 20 ml of DMF. The reaction was cooled to 0
°C in an ice bath while stirring. To this clear solution was added 0.9 ml of triethylsilane chloride
(TESCl) dropwise. The reaction was allowed to proceed for 20 minutes and monitored via TLC
(solvent: 1:1 hexanes; ethyl acetate, stain- H2SO4). Upon completion, the reaction was quenched
with saturated NH4Cl. The organic layer was extracted with ethyl acetate, washed two times with
brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford a slightly yellow
solid. Purification of this solid, dissolved in a small amount of DCM, was performed by column
chromatography on silica gel, with an increasing gradient of ethyl acetate in hexanes (10% - 40%
35
ethyl acetate in hexanes). Purification was monitored via TLC (solvent: 1:1 hexanes; ethyl acetate,
stain- H2SO4). Fractions 26-55 were collected and concentrated in vacuo to afford 4-I (959 mg,
80%) as a white solid. 1H NMR
10-(N,N’-dimethylcarbamoyl)-7-(triethylsilyl)-10-deacetylbaccatin III [4-II]:
To a 100 ml round-bottom flask under continual N2 purging was added 201 mg (0.305 mmol) of
4-I dissolved in 6 ml of THF. The solution was cooled to -40 °C in an acetone/dry ice bath while
stirring. To this mixture was added 0.35 ml (0.34 mmol) of LiHMDS, followed by the dropwise
addition of 40 μl (0.40 mmol) of N,N’-dimethylcarbamoyl chloride. The reaction was monitored
via TLC (solvent- 5% DCM in methanol, stain- H2SO4). During the course of the reaction, 1 eq.
of LiHMDS and 1 eq. of N,N’-dimethylcarbamoyl chloride was added at two separate time points
to promote the timely completion of the reaction. After 4 hours, the reaction was quenched with
saturated NH4Cl and diluted with water. The organic layer was extracted in ethyl acetate, washed
three times with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford
a slightly yellow solid. Purification of this solid, dissolved in a small amount of DCM, was
performed by column chromatography on silica gel with an increasing gradient of ethyl acetate in
hexanes (20% - 50% ethyl acetate in hexanes). Purification was monitored via TLC (solvent: 5%
DCM in methanol, stain- H2SO4). Fractions 16-24 were collected and concentrated in vacuo to
afford 4-II (202 mg, 89%) as a white solid. 10-(N,N’-dimethylcarbamoyl)-3’-dephenyl-3’-(2-
methylpropene-2-yl)-2’triisopropylsiilane-docetaxel [4-III]:
To a 100 ml round-bottom flask under continual N2 purging was added 202 mg (0.271 mmol) of
4-II and 201 mg (0.325 mmol) of 2-VIII dissolved in 7 ml of THF. The solution was cooled to 40
°C in an acetone/dry ice bath while stirring. To this mixture was added 0.38 ml (0.32 mmol) of
LiHMDS (dark orange color rather than characteristic clear color) dissolved in 1.0 ml of THF
dropwise. Upon completion, the reaction was diluted with water. The organic layer was extracted
with ethyl acetate, washed three times with brine, dried over anhydrous MgSO4, and concentrated
in vacuo to afford a crude, white solid. Purification of this solid, dissolved in a small amount of
DCM, was performed by column chromatography on silica gel with an increasing gradient of ethyl
acetate in hexanes (12% - 80 % ethyl acetate/hexanes). Purification was monitored via TLC
(solvent- 3:1 hexanes/ethyl acetate, stain- H2SO4). Fractions 13-28 were collected and
concentrated in vacuo to afford 4-III (192 mg, 64%; 97 mg of starting material 4-
36
II was recovered) as a white solid. 1H NMR (300 MHz, CDCl3) δ 0.57-0.63 (m, 6H), 0.83-0.97
(m, 9H), 1.02-1.10 (m, 21H), 1.24 (s, 3H), 1.34-1.39 (m, 1H), 1.40-1.49 (m, 15H), 1.69 (m, 3H),
1.75 (s, 3H), 1.79 (s, 3H), 1.92 (m, 1H), 2.05 (s, 3H), 2.36 (s, 3H), 2.40 (m, 1H), 2.94 (s, 3H),
3.06 (s, 3H), 3.85-3.88 (d, 1H, J = 7.2 Hz) 4.09-4.21 (m, 3H), 4.29-4.32 (d, 1H, J = 9.0 Hz),
4.43-4.44 (d, 1H, J = 3.0 Hz), 4.47 (m, 2H), 5.30-5.32 (d, 1H, J = 6.6 Hz), 5.68-5.70 (d, 1H, J =
6.0 Hz), 6.10 (m, 1H), 6.43 (s, 1H), 7.46 (t, 2H, J = 7.5 Hz), 7.60 (t, 1H, J = 7.5 Hz), 8.09-8.12 (d,
2H, J = 6.6 Hz). All data were found to be in agreement with literature values.
10-(N,N’-dimethylcarbamoyl)-3’-dephenyl-3’-(2-methylpropene-2-yl)docetaxel [SB-T-1216
(4-IV)]:
To a 100 ml round-bottom flask under continual N2 purging was added 192 mg (0.1703 mmol) of
4-III dissolved in a 1:1 mixture of acetonitrile:pyridine. The solution was allowed to stir and cool
to 0 °C in an ice bath. To this mixture was added 2 ml of HF/pyridine dropwise. The reaction was
allowed to proceed from 0 °C to room temperature overnight and monitored via TLC (solvent- 1:1
hexanes/ethyl acetate, stain- H2SO4). Upon completion, the reaction was diluted with water. The
organic layer was extracted three times with ethyl acetate, washed three times with CuSO4, two
times with water, three times with brine, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo to afford a slightly yellow solid. Purification of this solid, dissolved in DCM, was
performed by column chromatography on silica gel with an increasing gradient of ethyl acetate in
hexanes (40% - 80% ethyl acetate/hexanes). Purification was monitored via TLC (solvent- 3:1
hexanes/ethyl acetate, stain- H2SO4). Fractions 26-38 were collected and concentrated in vacuo to
afford an off-white solid. Further purification of this solid was performed by recrystalization in
ether anhydrous to afford SB-T-1216 (4-IV, 132 mg, 92%) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 1.24 (s, 3H), 1.30 (s, 3H), 1.36 (s, 9H), 1.42 (m, 1H), 1.66 (s, 3H), 1.70 (m, 1H), 1.76
(s, 6H), 1.91 (s, 4H), 2.04 (m, 1H), 2.35 (s, 3H), 2.50-2.56
(m, 1H), 2.95 (s, 3H), 3.04 (s, 3H), 3.22 (s, 1H), 3.45-3.48 (m, 1H), 3.80-3.82 (d, 1H, J = 7.0
Hz), 4.17-4.21 (m, 2H), 4.29-4.30 (d, 1H, J = 8.5 Hz), 4.43-4.46 (m, 1H), 4.74-4.75 (d, 1H, J =
7.5 Hz), 4.81-4.83 (d, 1H, J = 8.0 Hz), 4.96-4.981 (d, 1H, J = 9.5 Hz), 5.30-5.32 (d, 1H, J = 8.5
Hz), 5.65-5.66 (d, 1H, J =6.5 Hz), 6.16-6.19 (t, 1H, J = 9.0), 6.25 (s, 1H), 7.45-7.48 (t, 2H, J = 7.5
Hz), 7.58-7.61 (t, 1H, J = 7.5 Hz), 8.08-8.10 (d, 2H, J = 8.0 Hz). This material will be used for
37
further ongoing research efforts towards understanding the detailed mechanism of action of this
next generation taxoid.
§ 1.7 Summary
The β-LSM is an effective route towards the synthesis of new generation taxoids, which exhibit
greater efficacy against drug-resistant cell lines than their parent taxoids. Enantiopure βlactam can
be prepared in good yield via the Staudinger [2+2] ketene-imine cycloaddition, followed by
enzymatic resolution, and the chiral ester enolate-imine cyclocondensation. SB-T-
1216 is subsequently obtained in the ring opening coupling of this enantiopure β-lactam to a
modified baccatan, followed by deprotection. This material will be used for further ongoing
research efforts towards understanding the detailed mechanism of action of this next generation
taxoid.
§ 1.8 Acknowledgments
First, I’d like to extend my deepest gratitude to Dr. Iwao Ojima, Distinguished Professor of
Chemistry and Director of ICB&DD, for allowing me the opportunity to conduct research in his
lab. The support and constructive suggestions he provided has been a great help throughout my
time here. I’d also like to thank my mentors, Dr. Anushree Kamath and Jacob Vineberg, for
dedicating their time and skill towards helping me achieve the goals of my project. Their continual
guidance, support, encouragement, and advice have been greatly appreciated. Additional advice
and assistance provided by Edison S. Zuniga and Joshua Seitz was also valuable, and their help
has been greatly appreciated throughout the entirety of this project. This research was supported
by a grant from the National Cancer Institute.
§ 1.9 References
1. Siegel, R., Naishadham, D., Jemal, A., Cancer statistics, 2012. CA Cancer J Clin. 2012, 62, 10-
29.
38
2. Ford, H., Pardee, A. Cancer and the Cell Cycle. J. Cell. Biochem.. 1999, 75, 166-172.
3. Malumbres, M., Barbacid, M. Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev.
Cancer. 2009, 9, 153-166.
4. Ojima, I. Guided Molecular Missiles for Tumor-Targeting Chemotherapy-Case Studies Using
the Second-Generation Taxoids as Warheads. J. Med. Chem. 2008, 51, 3203-3221. 5. Rowinsky,
E., Donehower, R. Paclitaxel (Taxol). J. Med. 1995, 332, 1004-1014.
6. Ojima, I., Miller, M. Chemistry and Chemical Biology of Taxane Anticancer Agents. The
Chem. Rec. 2001, 1, 195-211.
7. Pollard, T., Earnshaw, W., Lippincott-Schwartz, J., Johnson, G. 2008. Cell Biology 2/e.
Elsevier Inc. 928 p.
8. Georg, G. I., Boge, T. C., Cheruvallath, Z. S., Clowers, J. S., Harriman, G. C. B., Hepperle,
M., Park, H. The medicinal chemistry of taxol. Taxol: Science and Applications, 1995, 317-375.
9. Schiff, P. B.; Fant, J.; Horwitz, S. B. Promotion of microtubule assembly in vitro by taxol.
Nature. 1979, 277, 665-667.
10. Schiff, P. B.; Horwitz, S. B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc.
Natl. Acad. Sci. 1980, 77, 1561-1565.
11. Zhao, Xianrui, Doctor of Philosophy Thesis, SUNY at Stony Brook, Stony Brook, NY.7. 12.
Cancer multidrug resistance. Nat. Biotechnol. 1999, 17, 94–95.
13. Higgins, CF., Linton, KJ. The ATP switch model for ABC transporters. Nat. Struct. Mol.
Biol., 2004, 11(10), 918-26.
14. Ojima, I., Slater, J., Michaud, E., Kuduk, S., Bounaud, P., Vrignaud, P., Bissery, M., Veith,
J., Pera, P., Bernacki, R. Syntheses and Structure-Activity Relationships of the SecondGeneration
Antitumor Taxoids: Exceptional Activity against Drug-Resistant Cancer Cells. J. Med. Chem.
1996, 39, 3889-3896. (was 13)
15. Ojima, I., Das, M. Recent Advances in the Chemistry and Biology of New Generation
Taxoids. J. Nat. Prod. 2009, 72, 554–565.
16. Ojima, I., Recent Advances in the β-Lactam Synthon Method. Acc. Chem. Res. 1995, 28,
383-389.
17. King, B., Sharpless, B. An efficient synthesis of enantiomerically pure trans-2
phenylcyclohexanol. Tetrahedron Lett. 1994, 35, 5611-5612.
39
18. J. Gonzalez, C. Aurigemma, L. Truesdale. Synthesis of (+)-(1S,2R)- and (−)-(1R,2S)-trans-
2Phenylcyclohexanol via Sharpless Asymmetric Dihydroxylation (AD). Org. Synth. 2002, 79, 93.
19. Sharpless, B., Kold, H., VanNieuwenhze, M. Catalytic Asymmetric Dihydroxylation. Chem.
Rev. 1994, 94, 2483-2547.
20. Ojima, I., Habus, I., Zhao, M., Zucco, M., Park, Y., Sun, C., Brigaud, T. New and Efficient
Approaches to the Semisynthesis of Taxol and its C-13 Chain Analogs by Means of the βLactam
Synthon Method. Tetrahedron. 1992, 4, 6985-7012.
21. Cossío, F.P., Arrieta, A., Sierra, M.A. The Mechanism of the Ketene−Imine (Staudinger)
Reaction in Its Centennial: Still an Unsolved Problem? Acc. Chem. Res., 2008, 41(8), 925–936.
22. Palomo, C., Jesus, M., Inaki, A., Oiarbide, G.M. Assymetric Synthesis of β-lactams by
Staudinger Ketene-Imine Cycloaddition Reactions. J. Org. Chem., 1999, 1999(12), 3223-3235.
23. Jiao, L., Liang, Y., Xu, J. Origin of the Relative Stereoselectivity of the β-Lactam
Formation in the Staudinger Reaction. J. Am. Chem. Soc., 2006, 128(18) 6060-6069.
24. Lynch, J.E., Riseman, S.M., Laswell, W.L., Volante, R.P., Smith, G.B., Shinkai, I.,
Tschaen, D.M. Mechanism of an acid chloride-imine reaction by low-temperature FT-IR: β -
lactam formation occurs exclusively through a ketene intermediate. J. Org. Chem., 1989, 54(16),
3792– 3796.
25. Lopez, R., Sordo, T.L, Sordo, J.A., Gonzalez, J. Torquoelectronic Effect in the Control of
the Stereoselectivity of Ketene-Imine Cycloaddition Reactions. J. Org. Chem., 1993, 58(25),
70367037.
26. Kovář, J., Ojima, I., Ehrlichová, M., Šmejkalová, B., Zanardi, I., Gut, I. Comparison of
Cell Death-inducing Effect of Novel Taxane SB-T-1216 and Paclitaxel in Breast Cancer Cells.
Anticancer Res., 2009, 29(8), 2951-2960.
27. Ojima, I., Jaracz, S., Chen, J., Kuznetsova, L. Recent advances in tumor-targeting
anticancer drug conjugates. Bioorg. Med. Chem., 2005, 13(2005), 5043–5054.
28. Ojima, I., Slater, J., Kuduk, S., Takeuchi, C., Gimi, R., Sun, C., Park, Y., Pera, P., Veith,
J., Bernacki, R. Syntheses and Structure-Activity Relationships of Taxoids Derived from 14-β-
Hydroxy-10-deacetylbaccatin III. J. Med. Chem. 1997, 40, 267-278.
40
29. Brieva, R., Crich, J.Z., Sih, C., Chemoenzymatic Synthesis of the C-13 Side Chain of
Taxol: Optically Active-3-Hydroxy-4-Phenyl β-lactam Derivatives. J. Org. Chem., 1993, 58,
10691075.