Discovery of 6-Diazo-5-oxo- l -norleucine (DON) Prodrugs with Enhanced CSF Delivery in Monkeys: A Potential Treatment for Glioblastoma

Article abstract of DOI:10.1021/acs.jmedchem.6b01069

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.

Full text of DOI:10.1021/acs.jmedchem.6b01069

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Article
Discovery of 6-Diazo-5-oxo-L-norleucine (DON) prodrugs with enhanced
CSF delivery in monkeys: a potential treatment for glioblastoma
Rana Rais, Andrej Jancarik, Lukas Tenora, Michael Nedelcovych, Jesse Alt, Judson Englert,
Camilo Rojas, Anne Le, Amira Elgogary, Jessica Tan, Lenka Monincova, Kelly Pate, Robert
J. Adams, Dana V. Ferraris, Jonathan D. Powell, Pavel Majer, and Barbara S. Slusher
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01069 • Publication Date (Web): 25 Aug 2016
Downloaded from http://pubs.acs.org on August 29, 2016
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Discovery of 6-Diazo-5-oxo-L-norleucine (DON) prodrugs
with enhanced CSF delivery in monkeys: a potential
treatment for glioblastoma
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Rana Rais , Andrej Jančařík , Lukáš Tenora , Michael Nedelcovych , Jesse Alt , Judson
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‡£
ξ,Ψ
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ξ
†
Englert , Camilo Rojas , Anne Le , Amira Elgogary , Jessica Tan , Lenka Monincová , Kelly
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£
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Ψ
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Pate , Robert Adams , Dana Ferraris , Jonathan Powell , Pavel Majer * Barbara S. Slusher *
‡
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¥
ξ
Ψ
Johns Hopkins Drug Discovery, Departments of Neurology, Pathology, Oncology, and
Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore,
Maryland 21205, U.S.A.
†
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic
v.v.i., Prague, Czech Republic
§
Department of Chemistry, McDaniel College, Westminster, Maryland, 21157, U.S.A.
#
These authors contributed equally
* Corresponding authors
KEYWORDS: glutamine antagonist, glutamine, 6ꢀDiazoꢀ5ꢀoxoꢀLꢀnorleucine, prodrug, brain,
cerebrospinal fluid, glioblastoma, pharmacokinetics,
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ABSTRACT
The glutamine antagonist 6ꢀdiazoꢀ5ꢀoxoꢀLꢀnorleucine (DON,1) has shown robust antiꢀcancer
efficacy in preclinical and clinical studies, but its development was halted due to marked
systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and
provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed.
To enhance DON’s therapeutic index, we utilized a prodrug strategy to increase its brain delivery
and limit systemic exposure. Unexpectedly, simple alkyl esterꢀbased prodrugs were ineffective
due to chemical instability cyclizing to form a unique diazoꢀimine. However masking both
DON’s amine and carboxylate functionalities imparted sufficient chemical stability for biological
testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several
provided excellent stability in monkey and human plasma. The most stable compound (5c,
methylꢀPOMꢀDONꢀisopropylꢀester) was evaluated in monkeys, where it achieved 10ꢀfold
enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to
DON utilization in GBM patients.
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INTRODUCTION
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Glioblastoma multiforme (GBM) is the most common and lethal form of glioma. Current
therapeutic options that extend survival rates in GBM patients after tumor resection are limited to
radiotherapy with concomitant administration of the DNAꢀalkylating agent such as
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temozolamide and/or the carmustine in a polymeric biodegradable wafer for controlled release.
But even under this standard of care, postoperative median survival rates approximate 14.6
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months, and average fiveꢀyear survival is less than 10%. There is, therefore, a significant
unmet medical need for more effective GBM treatments.
The ability of GBMs and other cancers to rapidly and persistently proliferate often outgrowing
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vascular supplies necessitates that they develop a specialized metabolism.
Glutamine
metabolism plays an essential role in this specialized metabolism by entering the TCA cycle and
then contributing to the biosynthetic pathways (nucleotide, protein and lipid synthesis) necessary
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for unchecked cell growth, rendering cancer cells dependent on glutamine. This so called
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“glutamine addiction” has been well characterized in GBMs
and other cancers including
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leukemia/lymphomas, lung, tripleꢀnegative breast cancer and pancreatic cancer.
To combat
this glutamine addiction, several groups have explored the utility of selective glutaminase
inhibitors.13, 14 Recently compounds that selectively target glutaminase (GLS1) have been
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developed
and one such compound is in clinical trials. However, the brain penetration of
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this GLS1 inhibitor is poor and so far the clinical efficacy of this approach has been modest.
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Accumulating evidence shows that, as opposed to selective inhibition of one glutamine utilizing
enzyme, broadly antagonizing glutamine utilization is a highly effective means of inhibiting
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tumor cell growth in vitro and in vivo.
6ꢀDiazoꢀ5ꢀoxoꢀLꢀnorleucine (DON), a nonꢀnatural
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amino acid with structural similarity to glutamine, was first isolated from Streptomyces bacteria
in the early 1950’s. Because of its reactive diazo group, DON has demonstrated the ability to
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alkylate several glutamineꢀutilizing enzymes such as glutaminase,
NAD synthase, and CTP
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synthetase and FGAR aminotransferase in the purine and pyrimidine biosynthetic pathways,
respectively. In preclinical models, DON robustly inhibited the growth of glutamineꢀdependent
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human cancer cells in vitro, and reduced tumor size and improved survival rates in vivo.
Because of the robust preclinical data, DON was evaluated in several clinical studies where it
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demonstrated promising results.
For example, DON administration caused >50% regression
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or stable disease in late stage adult patients and in children with hematological malignancies or
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solid tumors. Unfortunately, its development was hampered by dose limiting toxicities, many
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of which were GIꢀrelated
as the GI system is highly dependent on glutamine utilization.
One strategy to improve the therapeutic index of DON for GBM therapy would be to increase its
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brain exposure while limiting its systemic exposure and thus toxicity. The prodrug approach is
a wellꢀestablished strategy to alter the pharmacokinetic and tissue distribution of drug molecules.
With respect to DON, the most reactive functionality is its diazoketone, however derivatization
of the diazoketone functionality leads either to highly unstable compounds (e.g. ketals) or to
compounds that do not convert to DON (cyclic diazoꢀimine). Thus our prodrug strategy was
focused on prodrugging the derivatizable functionalities on DON, e.g. the carboxylate and the
amine. This was synthetically challenging because the addition of promoeities had to be
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performed under mild conditions to preserve the diazo ketone group. In addition, as detailed
below, our initial attempts to create simple alkyl ester prodrugs resulted in significant chemical
instability, making it critical to mask both DON’s carboxylate and amine functionalities to
achieve functional prodrugs.
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Herein we describe, for the first time, profound efficacy of DON in murine model of GBM,
although overt toxicities were observed. In attempt to increase DON’s therapeutic index, we
systematically synthesized several DON prodrugs. Our initial strategy involved masking DON’s
carboxylic acid with simple alkyl esters such as ethyl ester 2a and isopropyl ester 2b (Figure 1).
However, 2a-b exhibited chemical instability cyclizing to form a unique diazoꢀimine. Given this
instability, we next masked both the primary amine and the carboxylate of DON with prodrug
moieties. We utilized three types of amine promoieties including (oxodioxolenyl)methyl
carbamate esters (3a-b), dipeptides (4a-d), and pivaloylꢀoxyꢀmethyl (POM)ꢀbased esters (5a-c).
The dual promoietyꢀcontaining prodrugs resulted in sufficient chemical stability permitting
further evaluation in biological assays. While all of the prodrugs exhibited rapid metabolism in
mouse plasma precluding their comparative efficacy evaluation in mice, cross species
metabolism studies showed excellent plasma stability in monkeys and humans. Given this
similarity, we evaluated the most stable DON prodrug (5c, methylꢀPOMꢀDONꢀisopropylꢀester)
in pigꢀtail macaques where it achieved 10ꢀfold enhanced cerebrospinal fluid (CSF) to plasma
ratio versus DON, thus providing a possible clinical path to DON utilization in GBM patients.
CHEMISTRY
Scheme 1 outlines the synthesis and characterization of the ester based prodrugs 2a-b of DON
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and their subsequent cyclization to novel diazoꢀimines 9a-b. The pyroglutamate esters 6a-b
were FMOCꢀprotected to afford compounds 7a-b. Formation of the diazoketones 8a-b was
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accomplished using diazo(trimethylsilyl)methyllithium in good yield. Rapid deprotection with
piperidine afforded the ester based prodrugs 2a-b. Unexpectedly slow cyclization was observed
even under mild conditions (e.g. stirring in chloroform at room temperature) affording the novel
diazoꢀimines 9a and 9b. To our knowledge, this is the first example of this functional group
being described in the chemical literature. Attempts to avoid the cyclization of 2a-b by salt
formation to protonate the amine resulted in instability of the diazo group. Furthermore, unlike
most imines, the 5ꢀmember cyclic diazoꢀimines 9a-b were found to be stable, even at acidic pH,
and did not convert back to DON esters (results not shown).
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Given their sufficient chemical stability, compounds 2a and 2b were utilized as synthetic
intermediates for preparation of dual promoiety prodrugs (Schemes 2ꢀ4). The (oxodioxolenyl)
methyl carbamate adducts 3a-b were synthesized as outlined in Scheme 2 as previously
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described . The 4ꢀ(hydroxymethyl)ꢀ5ꢀmethylꢀ1,3ꢀdioxolꢀ2ꢀone 10 was reacted with Sꢀethyl
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carbonochloridothionate, followed by Nꢀhydroxysuccinimide to provide 11. Reaction of 2a-b
with 11 afforded the (oxodioxolenyl) methyl carbamate esters 3a-b in excellent yields.
Scheme 3 outlines the synthesis of DON dipeptide esters. DON esters 2a-b were coupled with
FmocꢀLꢀleucine or FmocꢀLꢀtryptophan using HBTU to form the protected dipeptides 12a-d.
Deprotection with diethyl amine or piperidine afforded the desired leucineꢀDON 4a-b and
tryptophanꢀDON prodrugs 4c-d.
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As shown in Scheme 4, the POM derivative 5a was synthesized from 2a using the POMꢀNꢀ
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hydroxysuccinimate ester in 40% yield. Introduction of methyl group in the POM ester led to
the formation of methylꢀPOM derivatives 5b and 5c, with an added chiral center. Both 5b and 5c
were synthesized from 2a-b using 1ꢀ((((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)oxy)carbonyl)oxy)ethyl
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pivalate . 5b was obtained as a mixture of two diastereomers (1:1 ratio) in 68% yield. 5c was
also obtained diastereomeric mixture (1:1) ratio, but was further purified into its corresponding
diastereomers 1 and 2, of which diastereomer 1 was used for subsequent biological testing as
described below.
RESULTS AND DISCUSSION
DON showed robust inhibition of glutamine metabolism and antitumor efficacy in a
murine GBM model
Despite several lines of evidence indicating the potential therapeutic efficacy of targeting
glutamine metabolism in GBM, the effect of DON on GBM tumor growth has not yet been
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reported in vivo. Using the U87 flank xenograft mouse model of GBM, , we first confirmed
that systemic administration of DON (0.8 mg/kg, i.p) inhibited glutamine metabolism as
reflected by an accumulation of endogenous glutamine in the tumor (Figure 2A; p <0.05) similar
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to other model systems.
We next evaluated its antitumor efficacy, and observed that DON
effectively halted tumor growth. Specifically, vehicleꢀtreated mice displayed significant tumor
growth over the course of the experiment, while DONꢀtreated mice (0.8 mg/kg, i.p, q.d.)
exhibited >50% reduction in tumor volume (Figure 2B; main effect of time [F(3,48) = 6.049, p =
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.0014]; treatment [F(1,16) = 33.42, p < 0.0001]; interaction [F(3,48) = 21.70, p < 0.0001]).
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Although DON exhibited excellent antiꢀtumor efficacy, all mice receiving DON displayed
significant signs of toxicity including weight loss (12 + 4.1%), hunching, ptosis, and lethargy.
These findings are consistent with other reports of DON’s efficacy and toxicity both in vitro and
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in vivo.
Simple DON alkyl ester prodrugs found to be unstable. Masking both DON’s carboxylate
and amine functionalities required to obtain stable prodrugs.
A prodrug strategy is often employed to enhance tissue penetration and change the
pharmacokinetic parameters of effective drugs. Indeed, prodrug strategies are common in drug
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development as 5–7% of the approved worldwide drugs are prodrugs. In the case of DON, the
reactive diazoketone functionality could not be derivatized since it led to either to highly
unstable compounds (e.g. ketals) or to compounds that did not convert to DON (e.g. cyclic
diazoꢀimine). Thus our strategy was to prodrug the derivatizable functionalities on DON, e.g. the
carboxylate and the amine. Initially, we masked the carboxylic acid with simple alkyl esters
such as ethyl 2a and isopropyl 2b. The synthesis of these two derivatives was straightforward
affording compounds in good yield. It is surprising to us that these simple DON alkyl esters had
not previously been reported in the chemical literature, given that DON chemistry and utility has
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been described by numerous groups for over 60 years.
discovered that 2a and 2b were unstable, slowly cyclizing to form unique diazoꢀimines 9a and
b. These two unique derivatives were found to be chemically stable even at acidic pH,
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precluding their use as DON prodrugs.
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carboxylate of DON with prodrug moieties. This dual promoiety strategy was rationalized to
eliminate the potential for cyclization and potentially further improve the lipophilicity. We
utilized three previously described amine promoieties including (oxodioxolenyl)methyl
carbamate esters (Figure 1, 3a-b), dipeptides (4a-d), and pivaloylꢀoxyꢀmethyl (POM)ꢀbased
esters (5a-c). These promoeities were chosen because they are reported to target distinct
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metabolic enzymes including paraoxonase , aminopeptidases , and carboxylesterases ,
respectively. To impart further metabolic stability of the POM derivative (Table 1, 5a), we
prepared corresponding methylꢀPOM analogs (5b, 5c). Unlike the simple carboxylic acid ester
DON prodrugs (e.g. 2b), the dual promoiety prodrugs exhibited sufficient chemical stability to
permit further evaluation (defined as >90% aqueous stability following 1 h incubation at pH 7.4).
All DON prodrugs were rapidly metabolized in mouse plasma, however 5b and 5c found to
be stable in human and monkey plasma
Table 1 outlines the plasma stability of DON prodrugs 3a-b, 4a-d and 5a-c. All prodrugs were
completely metabolized in mouse plasma within the 60 min incubation time. However in
monkey and human plasma, the prodrugs 5b and 5c, with methylꢀPOM on the amine and ethyl or
isopropyl ester on the carboxylate respectively, demonstrated moderate/high stability with 60ꢀ
75% of the prodrug remaining in monkey plasma, and 80ꢀ90% remaining in human plasma
within the 60 min incubation time. Given 5c had the best stability profile in human plasma, it
was selected for further evaluation in pharmacokinetic studies and compared to DON for its
ability to deliver to cerebrospinal fluid and liberate DON.
Lead prodrug 5c enhanced CSF delivery of DON in monkeys but not in mice
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As expected from a DON prodrug which is completely metabolized in mouse plasma, we found
that oral administration of DON (1) (0.8 mg/kg) and 5c at equivalent dose (1.8 mg/kg ) exhibited
similar DON plasma (Figure 3A) and brain (Figure 3B) concentration profiles when dosed in
mice. The AUC0ꢀt of DON following administration of DON and 5c in plasma were 1.25
nmol*h/mL and 1.22 nmol*h/mL respectively, suggesting rapid and complete liberation of DON
from 5c in vivo. Similarly in the mouse brain, the AUC0ꢀt of DON following DON or 5c
administration was 0.57 nmol*h/g and 0.69 nmol*h/g, respectively, with the brain/plasma
approximately 0.46 from DON vs 0.56 from prodrug 5c. These pharmacokinetic results
corroborated the in vitro metabolism studies suggesting 5c was rapidly converted to DON in
mouse plasma. Given this, the efficacy evaluation of 5c in mice would not be meaningful as it
would be similar to evaluating DON. Importantly, this conundrum is not uncommon in prodrug
discovery, where increased metabolism in rodents is well documented and higher species are
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necessary to mimic human metabolism.
Following the mouse studies, we evaluated the pharmacokinetics of DON and 5c (diastereomer
) in monkeys, as monkeys better mimicked the human plasma stability profile. In pigtail
1
macaques, i.v. administration of DON (1.6 mg/kg) and 5c (3.6 mg/kg; equivalent dose of DON)
demonstrated significantly different DON plasma profiles (Figure 4A). DON administration
provided high plasma exposures with AUC of 42.7 nmol*h/mL. In contrast, 5c administration
0
ꢀt
delivered ~ 7 fold lower plasma exposure of DON with AUC0ꢀt of 5.71 nmol*h/mL. The
opposite observation was seen in the CSF where enhanced DON levels were observed after 5c
administration. In the CSF at 30 min post dose, DON administration resulted in 0.33 nmol/g
DON while 5c delivered 1.43 nmol/g DON. When comparing plasma to CSF ratio at 30 min, 5c
demonstrated 10ꢀfold enhancement of DON CSF delivery versus DON (Figure 4B).
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CONCLUSION
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We demonstrate profound efficacy of DON in a murine model of GBM, although overt toxicity
was observed. To enhance DON’s therapeutic index, we utilized a prodrug strategy to increase
its brain delivery and limit its systemic exposure. While our dual promoeity prodrugs exhibited
rapid metabolism in mice, our lead prodrug 5c provided excellent stability in human and monkey
plasma, and achieved a 10ꢀfold enhanced CSF:plasma ratio versus DON in monkeys. This
strategy may provide a path to DON utilization in GBM patients.
EXPERIMENTAL SECTION
The commercially available HPLC grade methanol, catalysts and reagent grade materials were
used as received. TLC was performed on Silica gel 60 F254‐coated aluminum sheets (Merck)
and
spots were detected by the solution of Ce(SO ) . 4H O (1%) and H P(Mo O ) (2%) in sulfuric
4
2
2
3
3
10 4
acid (10%). Flash chromatography was performed on Silica gel 60 (0.040‐0.063 mm, Fluka) or
on Biotage® KP‐C18‐HS or KP‐Sil® SNAP cartridges using the Isolera One HPFC system
(Biotage, Inc.). All chemicals were purchased from SigmaꢀAldrich and were used without further
1
13
purification. The H NMR spectra were measured at 400.1 MHz, 500.1 MHz or 600.1 MHz, C
1
NMR spectra at 100.8 MHz, 125.7 MHz or 150.9 MHz. For standardization of H NMR spectra
the internal signal of TMS (δ 0.0, CDCl ) or residual signals of solvents (δ 7.26 for CDCl , δ
3
3
1
3
2
.05 for CD COCD and δ 3.31 for CD OD) were used. In the case of C spectra the residual
3
3
3
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signals of solvents (δ 77.00 for CDCl , δ 29.84 and δ 206.26 for CD COCD and δ 49.00 for
3
3
3
CD OD) were used. The chemical shifts are given in δ‐scale, the coupling constants J are given
3
in Hz. The ESI mass spectra were recorded using ZQ micromass mass spectrometer (Waters)
equipped with an ESCi multimode ion source and controlled by MassLynx software.
Alternatively, the low resolution ESI mass spectra were recorded using a quadrupole orthogonal
acceleration time‐of‐flight tandem mass spectrometer (Q‐Tof micro, Waters) and high resolution
ESI mass spectra using a hybrid FT mass spectrometer combining a linear ion trap MS and the
Orbitrap mass analyzer (LTQ Orbitrap XL, Thermo Fisher Scientific). The conditions were
optimized for suitable ionization in the ESI Orbitrap source (sheat gas flow rate 35 a.u., aux gas
flow rate 10 a.u. of nitrogen, source voltage 4.3 kV, capillary voltage 40 V, capillary temperature
0
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0
275 °C, tube lens voltage 155 V). The samples were dissolved in methanol and applied by direct
injection. Optical rotations were measured in CHCl or DMF using an Autopol IV instrument
3
(Rudolph Research Analytical). The IR spectra were measured in CHCl or KBr. Purity for each
3
compound was established using HPLC (Jasco Inc.) equipped with a Reprosil 100 C18, 5 ꢀm,
250 x 4 mm column. The analysis was performed using a gradient of 2% CH CN/98%:H O with
3 2
0
.1% TFA ꢀ 100% CH CN, with UV detection, λ=210 nm. All compounds were deemed >95%
3
pure by this method and lead compound 5c was >99% pure.
Ethyl 2-amino-6-diazo-5-oxohexanoate (2a). Compound 8a (1.00 g, 2.37 mmol, 1 eq.) was
dissolved in dry CH Cl (10 mL). Piperidine (505 mg, 579 ꢁL, 5.93 mmol, 2.5 eq.) was added
2
2
and the reaction mixture was stirred at room temperature (rt) for 4 h. The organic solvent was
evaporated in vacuo. The residue was chromatographed on silica gel (CHCl /methanol 30:1) to
3
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afford the desired product (312 mg, 66%) as a yellow oil. H NMR (CDCl ): δ 1.27 (3H, t, J =
3
7.1), 1.55 (2H, bs), 1.78 – 1.88 (1H, m), 2.06 – 2.17 (1H, m), 2.40 – 2.54 (2H, m), 3.44 (1H, dd,
13
J = 8.3, 5.1), 4.17 (2H, q, J = 7.1), 5.27 (1H, s); C NMR (101 MHz, CDCl ): 14.24, 29.56,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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49
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59
60
3
6.86, 53.77, 54.56, 61.05, 175.58, 194.15; IR (CHCl ): 3410 w, vbr, 3327, vw, vbr, 2986 m,
3
2941 w, 2910 w, 2874 w, 2110 s, 1739 vs, 1641 m, 1605 m, sh, 1586 m, 1552 w, 1513 w, br,
ꢀ1
1
476 w, 1463 m, 1446 m, 1395 m, sh, 1377 s, 1200 s, 1115 m, 1096 m cm ; ESI MS: 182 ([M –
+
H O + H] ); HR ESI MS: calcd for C H N O 182.0930; found 182.0931.
2
12 12
3
2
Isopropyl 2-amino-6-diazo-5-oxohexanoate (2b). The FMOCꢀprotected compound 8b (900
mg, 2.07 mmol, 1 eq.) was dissolved in dry CH Cl (10 mL). Piperidine (514 ꢁL, 5,17 mmol, 2.5
2
2
eq.) was added and the reaction mixture was stirred at rt for 4 h. The organic solvent was
evaporated in vacuo. The residue was chromatographed on silica gel (CHCl /methanol 30:1) to
3
1
afford the desired product (290 mg, 66%) as a yellow oil. H NMR (CDCl ): δ 1.21 (3H, d, J =
3
1.8), 1.23 (3H, d, J = 1.8), 1.52 (2H, bs), 1.74 – 1.85 (1H, m), 2.02 – 2.10 (1H, m), 2.36 – 2.53
1
3
(2H, m), 3.37 (1H, dd, J = 8.4, 5.0), 5.00 (1H, hept, J = 6.3), 5.27 (1H, s); C NMR (101 MHz,
CDCl ): 21.87, 21.89, 29.65, 36.99, 53.94, 54.66, 68.64, 175.21, 194.25; Optical rotation:
3
2
2
[
α] + 6.5° (c 0.444, CHCl ); IR (CHCl ): 3390 w, 3323 vw, 3116 w, 2984 s, 2939 m, 2877 w,
D 3 3
2109 vs, 1725 vs, 1640 s, 1467 m, 1454 m, 1439 w, sh, 1388 s, sh, 1376 vs, 1349 s, 1199 s, 1106
ꢀ1
+
vs cm ; ESI MS: 236 ([M + Na] ); HR ESI MS: calcd for C H O N Na 236.1006; found
9
15
3
3
236.1007.
Ethyl-6-diazo-2-((((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)amino)-5-
2
oxohexanoate (3a). NHS ester 11 (180 mg, 0.663 mmol, 1.1 eq) was suspended in dry CH Cl2
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(
5 mL). The reaction mixture was cooled to 0 °C and compound 2a (120 mg, 0.602 mmol, 1 eq.)
in dry CH Cl (3 mL) was added dropwise. The mixture was stirred for 15 minutes at 0 °C and
2
2
then 1 h at rt. The crude product was purified by column chromatography (EtOAc/hexane 1:1)
0
1
2
3
4
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8
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1
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1
and the desired compound 3a was obtained in 65% yield (139 mg) as a yellow oil. H NMR
(CDCl ): δ 1.27 (3H, t, J = 7.1), 1.95 – 2.06 (1H, m), 2.10 – 2.27 (1H, m), 2.16 (3H, s), 2.31 –
3
2
.50 (2H, m), 4.19 (2H, q, J = 7.1), 4.29 (1H, td, J = 8.1, 4.8), 4.82 (2H, dd, J = 13.9), 5.29 (1H,
1
3
bs), 5.72 (1H, d, J = 7.7); C NMR (CDCl ): 9.48, 14.24, 27.18, 36.32, 53.76, 54.58, 55.00,
3
2
2
6
1.92, 133.90, 140.08, 152.30, 155.26, 171.63, 193.52; Optical rotation: [α] + 12.2° (c 0.229,
D
CHCl ); IR (CHCl ): 3425 m, 3345 w, vbr, 3116 w, 2111 vs, 1838 s, sh, 1820 vs, 1736 vs, 1722
3
3
ꢀ1
+
vs, sh, 1640 s, 1511 s, 1229 s, 1200 s, 1114 m, 1097 m, 1045 s, cm ; ESI MS: 378 ([M + Na] );
HR ESI MS: calcd for C H O N Na 378.09079; found 378.09102.
1
4
17
8
3
Isopropyl 6-diazo-2-((((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)amino)-5-
oxohexanoate (3b). Compound 11 (70 mg, 0.258 mmol, 1.1 eq.) was dissolved in CH Cl (2
2
2
mL). This solution was cooled to 0 °C and a solution of the compound 2b (50 mg, 0.235 mmol, 1
eq.) in CH Cl (1 mL) was added dropwise. The reaction mixture was stirred for 15 min at 0 °C
2
2
and the cooling bath was removed. The reaction mixture was then stirred at rt for 1 h. The
organic solvent was evaporated in vacuo. The residue was chromatographed on silica gel
1
(chloroform/acetone 10:1) to afford the desired product (72 mg, 83%) as a yellow oil. H NMR
(CDCl ): δ 1.24 (3H, d, J = 3.4), 1.26 (3H, d, J = 5.6), 1.94 – 2.04 (1H, m), 2.13 – 2.24 (1H, m),
3
2.16 (3H, s), 2.32 – 2.49 (2H, m), 4.23 – 4.28 (1H, m), 4.77 – 4.87 (2H, m), 5.04 (1H, hept, J =
13
6
.3), 5.28 (1H, s), 5.66 (1H, d, J = 7.9); C NMR (101 MHz, CDCl ): 9.51, 21.82, 21.85, 27.32,
3
3
6.33, 53.86, 54.57, 69.79, 133.93, 140.07, 152.31, 155.25, 171.12, 193.50; Optical rotation:
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[
α] + 15.1° (c 0.417, CHCl ); IR (CHCl ): 3424 w, 3012 w, 2984 w, 2935 w, 2111 s, 1836 sh,
D 3 3
s, 1821 vs, 1736 sh, vs, 1725 vs, 1641 m, 1603 sh, w, 1509 s, 1391 sh, m, 1383 s, 1366 sh, m,
ꢀ1
+
1
105 s cm ; ESI MS: 392 ([M + Na] ); HR ESI MS: calcd for C H O N Na 392.10644; found
15 19 8 3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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3
92.10650.
Ethyl 2-(2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoate (4a). Compound 12a (945
mg, 1.77 mmol, 1 eq.) was dissolved in dry CH Cl (9 mL). Diethylamine (646 mg, 914 µL, 8.84
2
2
mmol, 5 eq.) was added by syringe. The reaction mixture was stirred for 4 h at rt under inert
atmosphere followed by evaporation of solvent. The crude product was purified by column
chromatography (CHCl /MeOH 15:1, R 0.38) and a yellowish amorphous solid (500 mg) was
3
f
1
obtained in 91% yield. H NMR (CDCl ): δ 0.94 (6H, dd, J = 14.0, 6.3), 1.27 (3H, t, J = 7.1),
3
1.28 – 1.37 (1H, m), 1.45 (2H, s), 1.57 – 1.83 (2H, m), 1.92 – 2.08 (1H, m), 2.16 – 2.26 (1H, m),
2.26 – 2.49 (2H, m), 3.39 (1H, dd, J = 10.0, 3.9), 4.19 (2H, dq, J = 7.1, 1.4), 4.54 (1H, dt, J =
13
8.5, 4.8), 5.30 (1H, s), 7.85 (1H, d, J = 8.3); C NMR (CDCl ): 14.25, 21.40, 23.54, 24.97,
3
22
27.71, 36.82, 44.29, 51.48, 53.60, 54.86, 61.65, 171.99, 175.99, 193.67; Optical rotation: [α]
D
ꢀ 54.8° (c 0.323, CHCl ); IR (KBr): 3433 w, 3317 w, 3204 m, 3204 w, 3074 m, 2959 m, 2872 m,
3
ꢀ1
2111 m, 1733 w, sh, 1678 s, 1654 s, sh, 1630 m, 1509 w, 1387 m, 1348 m, cm ; ESI MS: 335
+
([M + Na] ); HR ESI MS: calcd for C H O N 313.18703; found 313.18712.
14 25
4
4
Isopropyl 2-(2-amino-4-methylpentanamido)-6-diazo-5-oxohexanoate (4b). The same
method of preparation was used as for compound 4a. Compound 12b (1.75 g, 3.19 mmol, 1 eq.)
was dissolved in CH Cl (20 mL). Piperidine (788 ꢁL, 7.98 mmol, 2.5 eq.) was added and the
2
2
mixture was stirred for 4 h at rt. The solvent was evaporated and the crude mixture was purified
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on silica gel (CHCl /MeOH 20:1). The product 4b (660 mg, 63%) was obtained as a yellow,
3
1
amorphous solid. H NMR (CDCl ): δ 0.94 (3H, d, J = 6.3), 0.98 (3H, d, J = 6.4), 1.25 (3H, d, J
3
=
–
2.8), 1.26 (3H, d, J = 2.8), 1.35 – 1.42 (1H, m), 1.66 – 1.79 (4H, m), 1.95 – 2.04 (1H, m), 2.17
2.25 (1H, m), 2.33 – 2.49 (2H, m), 3.48 (1H, dd, J = 9.7, 4.1), 4.52 (1H, td, J = 8.5, 4.7), 5.04
0
1
2
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7
8
9
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1
2
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8
9
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2
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4
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7
8
9
0
1
2
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4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
(1H, hept, J = 6.3), 5.35 (1H, s), 7.87 (1H, d, J = 8.3); C NMR (CDCl ): 21.43, 21.83 (2C),
3
23.53, 24.97, 27.82, 36.85, 44.26, 51.59, 53.61, 69.42, 171.48, 175.87, 193.76; Optical rotation:
2
2
[
α] + 7.0° (c 0.487, CHCl ); IR (CHCl ): 3412 w, 3343 w, 2110 vs, 1731 s, 1663 s, 1643 sh, s,
D 3 3
ꢀ1
1
603 sh, w, 1510 s, 1413 w, 1386 sh, s, 1376 sh, s, 1370 sh, s, 1349 m, 1145 m, 1105 s cm ; ESI
+
MS: 327 ([M + H] ); HR ESI MS: calcd for C H O N Na 327.20268; found 327.20280.
1
5
27
4
4
Ethyl 2-(2-amino-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate (4c). Compound
2c (303 mg, 0.499 mmol, 1 eq.) was dissolved in dry CH Cl (5 mL). Piperidine (212 mg, 244
1
2
2
µL, 2.49 mmol, 5 eq.) was added by syringe. The reaction mixture was stirred for 4.5 h at rt
under inert atmosphere followed by evaporation of solvent. The crude product was purified by
column LC (CHCl /MeOH 20:1 to 10:1, R 0.29) and a colorless solid (96 mg) was obtained in
3
f
1
5
0% yield. H NMR (CDCl ): δ 1.27 (3H, t, J = 7.0), 1.57 (2H, bs), 1.91 – 2.01 (1H, m), 2.06 –
3
2.27 (3H, m), 3.05 (1H, dd, J = 14.4, 8.1), 3.30 (1H, dd, J = 14.4, 4.0), 3.75 (1H, dd, J = 8.1,
4.2), 4.18 (2H, qd, J = 7.1, 2.0), 4.55 (1H, td, J = 8.3, 4.1), 5.11 (1H, bs), 7.09 (1H, d, J = 2.4),
.12 (1H, ddd, J = 8.1, 7.0, 1.1), 7.20 (1H, ddd, J = 8.1, 7.0, 1.2), 7.37 (1H, dt, J = 8.2, 1.0), 7.68
7
13
(
1H, dd, J = 7.9, 1.1), 7.88 (1H, d, J = 8.3), 8.31 (1H, bs); C NMR (101 MHz, CDCl ): 14.29,
3
27.60, 30.82, 36.58, 51.56, 54.77, 55.50, 61.71, 111.36, 111.49, 119.23, 119.79, 122.39, 123.45,
22
127.69, 136.52, 171.92, 175.02, 193.81; Optical rotation: [α]
ꢀ 57.6° (c 0.210, DMF); IR
D
(
CHCl ): 3479 m, 3355 w, 3215 vw, 3116 w, 2982 m, 2930 m, 2872 w, 2855 w, 2110 vs, 1737 s,
3
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ꢀ1
1
687 s, 1641 m, 1511 m, 1373 m, 1353 m, 1336 m, sh, 1191 m, 1115 w, 1092 m, 1009 w, cm ;
+
ESI MS: 408 ([M + Na] ); HR ESI MS: calcd for C H O N Na 408.16423; found 408.16435.
19 23
4
5
10
11
12
13
14
15
16
17
18
19
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Isopropyl
2-(2-amino-3-(1H-indol-3-yl)propanamido)-6-diazo-5-oxohexanoate
(4d).
Compound 12d (500 mg, 0.804 mmol, 1 eq.) was dissolved in dry CH Cl (15 mL). Piperidine
2
2
(342 mg, 393 µL, 4.02 mmol, 5 eq.) was added by syringe. The reaction mixture was stirred for 4
h at rt under inert atmosphere. DCM was evaporated. The crude product was purified by column
chromatography (CHCl /MeOH 30:1, R 0.14) and yellow amorphous solid (170 mg) was
3
f
1
obtained in 53 % yield. H NMR (CDCl ): δ 1.24 (3H, d, J = 6.0), 1.25 (3H, d, J = 6.0), 1.50
3
(2H, bs), 1.88 – 2.00 (1H, m), 2.04 – 2.27 (3H, m), 3.04 (1H, dd, J = 14.4, 8.2), 3.30 (1H, ddd, J
=
14.2, 4.2, 0.9), 3.74 (1H, dd, J = 8.1, 4.2), 4.51 (1H, td, J = 8.3, 4.0), 5.02 (1H, hept, J = 6.0),
5.11 (1H, bs), 7.08 (1H, d, J = 2.3), 7.11 (1H, ddd, J = 8.0, 7.1, 1.0), 7.19 (1H, ddd, J = 8.1, 7.1,
1
3
1.1), 7.36 (1H, d, J = 8.1), 7.67 (1H, d, J = 7.9), 7.88 (1H, d, J = 8.2), 8.45 (1H, bs); C NMR
(
CDCl ): 21.83, 27.65, 30.81, 36.55, 51.64, 54.80, 55.51, 69.49, 111.37 (2C), 119.17, 119.72,
3
22
122.31, 123.48, 127.67, 136.53, 171.42, 175.09, 193.92; Optical rotation: [α] ꢀ 1.2° (c 0.012,
D
CHCl ); IR (CHCl ): 3311 m, vbr, 2980 w, 2924 w, 2874 w, vs, 2853 w, 2104 vs, 1731 s, 1650
3
3
s, br, 1618 m, sh, 1512 m, 1388 m, sh, 1375 s, 1253 m, 1232 m, 1183 m, 1145 m, 1105 s, 1010
ꢀ1
+
w, 972 vw, 933 vw, 744 m, cm ; ESI MS: 422 ([M + Na] ); HR ESI MS: calcd for
C H O N Na 422.17988; found 422.17992.
20
25
4
5
Isopropyl 6-diazo-5-oxo-2-((((pivaloyloxy)methoxy)carbonyl)amino)hexanoate (5a). The Nꢀ
hydroxysuccinimate POM ester (320 mg, 1.17 mmol, 1.0 eq.) was suspended in dry CH Cl (6
2
2
mL). The reaction mixture was cooled to 0 °C and 2b (250 mg, 1.17 mmol, 1 eq.) in dry CH Cl2
2
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(
3 mL) was added dropwise. The mixture was stirred for 15 minutes at 0 °C and then 2 h at rt.
The crude product was purified by column chromatography (EtOAc/hexane 1:2, R 0.21) and the
f
1
desired compound 5a was obtained in 40% yield (175 mg) as a yellow oil. H NMR (400 MHz,
0
1
2
3
4
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7
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9
0
1
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9
0
CDCl ): δ 1.19 (9H, s), 1.23 (3H, d, J = 6.2), 1.24 (3H, d, J = 6.2), 1.90 – 2.05 (1H, m), 2.14 –
3
2.25 (1H, m), 2.31 – 2.51 (2H, m), 4.28 (1H, td, J = 8.2, 4.7), 5.03 (1H, hept, J = 6.2), 5.27 (1H,
13
bs), 5.65 (1H, d, J = 8.1), 5.69 (1H, d, J = 5.7), 5.73 (1H, d, J = 5.7); C NMR (101 MHz,
CDCl ): 21.79, 21.81, 26.97 (3C), 36.34, 38.86, 53.63, 54.90, 69.75, 80.33, 154.40, 171.01,
3
22
1
77.51, 193.43; Optical rotation: [α] + 13.0° (c 0.184, CHCl ); IR (CHCl ): 3424 m, 3354 w,
D 3 3
br, 3116 w, 2984 s, 2937 m, 2875 s, 2110 vs, 1747 vs, 1730 vs, sh, 1642 s, 1512 s, 1481 m, 1466
ꢀ1
m, 1453 m, 1377 s, 1282 s, 1182 m, 1145 s, 1105 s, 994 s, 942 m, cm ; ESI MS: 394 ([M +
+
Na] ); HR ESI MS: calcd for C H O N Na 394.15847; found 394.15855.
1
6
25
7
3
Ethyl 6-diazo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)-5-oxohexanoate (5b). NHS ester
ꢀ((((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)oxy)carbonyl)oxy)ethyl pivalate (238 mg, 0.828 mmol, 1.1 eq)
was suspended in dry CH Cl (6 mL). The reaction mixture was cooled to 0 °C and compound
1
2
2
2a (150 mg, 0.753 mmol, 1 eq.) in dry CH Cl (3 mL) was added by drop wise. The mixture was
2
2
stirred for 15 minutes at 0 °C and then 1 h at rt. The crude product was purified by column
chromatography (EtOAc/hexane 1:10 to 1:2) and the desired compound 5b was obtained in 68 %
yield (190 mg) as an yellow oil (mixture of isomers 1:1). Splitting of signals is due to presence
1
of diastereoisomers. H NMR (400 MHz, CDCl ): 1.16 1.18 (9H, s), 1.26 (3H, t, J = 7.1), 1.43
3
1.45 (3H, s), 1.88 – 2.05 (1H, m), 2.14 – 2.26 (1H, m), 2.30 – 2.50 (2H, m), 4.18 (2H, q, J = 7.1),
1
3
4
.24 – 4.34 (1H, m), 5.31 5.33 (1H, bs), 5.51 5.55 (1H, d, J = 7.7), 6.74 6.76 (1H, q, J = 7.3). C
NMR (101 MHz, CDCl ): 14.23 14.24, 19.72 19.74, 26.95 26.97, 27.57 27.80, 36.31 36.46,
3
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8.74 38.75, 53.33 53.48, 54.89 54.89, 61.85 61.87, 89.66 89.81, 153.87 153.95, 171.61 171.77,
2
2
76.56 176.68, 193.56 193.75. Optical rotation: [α] + 16.2° (c 0.259, CHCl ). IR (CHCl3):
D
3
428 m, 3358 w, 3116 w, 2982 s, 2874 m, 2110 s, 1740 vs, 1640 s, 1510 s, 1480 m, 1393 s (sh),
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57
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59
60
ꢀ1
+
377 s, 1349 s, 1284 s, 1232 s, 1025 s, cm . ESI MS: 394 ([M + Na] ). HR ESI MS: calcd for
C H O N Na 394.15847; found 394.15886.
16
25
7
3
Isopropyl 6-diazo-5-oxo-2-(((1-(pivaloyloxy)ethoxy)carbonyl)amino)hexanoate (5c).
solution of 1ꢀ((((2,5ꢀdioxopyrrolidinꢀ1ꢀyl)oxy)carbonyl)oxy)ethyl pivalate (297 mg, 1.032 mmol,
.1 eq.) was made in dry CH Cl (8 mL) and the mixture was cooled to 0 °C. Compound 2b (200
A
1
2
2
mg, 0.938 mmol) dissolved in dry CH Cl (4 mL) was added by syringe over 5 minutes. The
2
2
reaction mixture was spontaneously heated to rt and stirred for further 2 h. The CH Cl was
2
2
evaporated. The crude product was purified by column chromatography (CHCl /acetone 20:1) to
3
afford two diastereomers (90 mg, R = 0.25 and 50 mg, R = 0.2, 39%) as yellow oils. The NMR
f
f
1
spectra of both diastereomers were undistinguishable at 400 MHz resolution. H NMR (400
MHz, CDCl ): δ 1.18 (9H, s), 1.25 (3H, d, J = 3.0), 1.26 (3H, d, J = 3.1), 1.46 (3H, d, J = 5.4),
3
1.94 – 2.04 (1H, m), 2.15 – 2.24 (1H, m), 2.31 – 2.49 (2H, m), 4.24 – 4.30 (1H, m), 5.04 (1H,
13
hept, J = 6.2), 5.28 (1H, s), 5.50 (1H, d, J = 7.9), 6.77 (1H, q, J = 5.4); C NMR (101 MHz,
CDCl ): 19.78, 21.83, 21.85, 27.00 (3C), 27.79, 36.56, 38.79, 53.58, 69.76, 89.70, 153.90,
3
171.16, 176.61, 193.59; IR (CHCl ): 3427 w, 3116 w, 2984 m, 2960 sh, m, 2937 w, 2875 w,
3
2857 sh, w, 2110 s, 1745 sh, vs, 1730 sh, vs, 1641 m, 1508 s, 1480 m, 1467 sh, w, 1461 w, 1455
ꢀ
1
sh, w, 1392 sh, s, 1383 sh, s, 1377 s, 1371 sh, s, 1365 sh, s, 1350 m, 1027 m cm . Diastereomer
22
+
1
: Optical rotation: [α] + 22.0° (c 0.191, CHCl ); ESI MS: 408 ([M + Na] ); HR ESI MS:
D 3
calcd for C H O N Na 408.17412; found 408.17425. Diastereomer 2: Optical rotation:
1
7
27
7
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22
+
[
α]
+ 7.6° (c 0.158, CHCl ); ESI MS: 408 ([M + Na] ); HR ESI MS: calcd for
D 3
C H O N Na 408.17412; found 408.17421. Diastereomer 1 was used for all biological studies.
17 27
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-(9H-Fluoren-9-ylmethyl)-2-ethyl-5-oxopyrrolidine-1,2-dicarboxylate (7a). Ethyl 5ꢀ
oxopyrrolidineꢀ2ꢀcarboxylate 6a (4.00 g, 25.45 mmol, 1 eq.) was dissolved in dry THF (120 mL)
under inert atmosphere and cooled to ꢀ78 °C. A solution of LiHMDS (1M in THF, 24.2 mL,
2
4.18 mmol, 0.95 eq.) was added dropwise and the solution was stirred at this temperature for 20
min. The resultant yellow mixture was transferred via cannula to a solution of FMOCꢀchloride
32.9 g, 127.3 mmol, 5 eq.) in dry THF (90 mL) at ꢀ78 °C. The reaction mixture was stirred at ꢀ
8 °C for 2 h and at rt overnight (18 h). After this period the reaction was quenched with
(
7
saturated NH Cl (34 mL) and water (18 mL). Water phase was extracted with EtOAc (60 mL)
4
and combined organics were washed with brine (2×100 mL) and dried over anhydrous MgSO4.
The organic solvent was evaporated in vacuo. The residue was chromatographed on silica gel
(hexane/EtOAc 3:1 to 1:1) and finally on reverse phase HPLC (MeOH/H O 2:1 to MeOH) to
2
1
afford the desired product 7a (8.40 g, 87%) as a colorless solid. H NMR (CDCl ): δ 1.26 (3H,
3
d, J = 7.1), 2.12 (1H, ddt, J = 13.4, 9.4, 2.9), 2.40 (1H, ddt, J = 13.4, 10.8, 9.3), 2.57 (1H, ddd, J
=
17.5, 9.2, 3.1), 2.72 (1H, ddd, J = 17.5, 10.7, 9.4), 4.20 (2H, q, J = 7.1), 4.30 (1H, t, J = 7.3),
4
.44 (1H, dd, J = 10.5, 7.4), 4.57 (1H, dd, J = 10.6, 7.3), 4.65 (1H, dd, J = 9.4, 2.5), 7.33 (2H, tt,
J = 7.4, 1.1), 7.41 (2H, tdd, J = 6.9, 1.3, 0.6), 7.71 (1H, dd, J = 7.5, 1.0), 7.75 (1H, dd, J = 7.5,
13
1
6
1
3
.0), 7.77 (2H, dd, J = 7.8, 1.0); C NMR (CDCl ): 14.14, 21.96, 31.21, 46.62, 58.76, 61.92,
3
9.03, 119.99, 120.01, 125.30, 125.44, 127.26 (2C), 127.91 (2C), 141.27, 141.29, 143.35,
2
2
43.40, 151.44, 170.80, 172.99; Optical rotation: [α] ꢀ 17.2° (c 0.285, CHCl ); IR (CHCl ):
D
3
3
068 m, 2985 m, 2941 w, 2898 w, 2875 w, 1797 vs, 1745 vs, br, 1723 vs, 1609 w, 1580 vw,
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478 m, 1463 m, 1452 s, 1400 m, sh, 1385 s, 1197 vs, 1116 vw, sh, 1104 m, 1097 w, sh, 1033 s,
ꢀ1
+
21 m, 426 w, cm ; ESI MS: 402 ([M + Na] ); HR ESI MS: calcd for C H O NNa
22 21
5
02.13119; found 402.13127.
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1-((9H-Fluoren-9-yl)methyl)-2-isopropyl)-5-oxopyrrolidine-1,2-dicarboxylate (7b). The 1ꢀ
methylethyl 5ꢀoxoprolinate 6b (2.94 g, 17.16 mmol, 1 eq.) was dissolved in dry THF (90 mL)
under argon and cooled to ꢀ78 °C. A solution of LiHMDS (1M in hexane, 16.3 mL, 16.30 mmol,
0.95 eq.) was added dropwise and the solution was stirred at the same temperature for 15 min.
The resultant yellow mixture was transferred via cannula to a solution of FMOCꢀchloride (22.2
g, 85.8 mmol, 5 eq.) in dry THF (90 mL) at ꢀ78 °C. The reaction mixture was stirred at ꢀ78 °C
for 2 h. After this period the reaction was quenched with saturated NH Cl (100 ml). Then it was
4
extracted with ethyl acetate (3×50 mL), the combined organic layers were washed with water (40
mL), brine (40 mL) and dried over anhydrous MgSO . The organic solvent was evaporated in
4
vacuo. The residue was chromatographed on silica gel (hexane/EtOAc 2:1) to afford the desired
1
product 7b (6.2 g, 92%) as a colorless solid. H NMR (CDCl ): δ 1.23 (3H, d, J = 6.2), 1.26 (3H,
3
d, J = 6.3), 2.06 – 2.13 (1H, m), 2.34 – 2.45 (1H, m), 2.53 – 2.61 (1H, m), 2.67 – 7.76 (1H, m),
4.31 (1H, t, J = 7.5), 4.40 – 4.44 (1H, m), 4.53 – 4.57 (1H, m), 4.65 (1H, dd, J = 9.4, 2.6), 5.07
1
3
(
1H, hept, J = 6.3), 7.31 – 7.35 (2H, m), 7.39 – 7.43 (2H, m), 7.71 – 7.78 (4H, m); C NMR
(
CDCl ): 21.69, 21.78, 22.01, 31.31, 46.64, 58.99, 69.20, 69.78, 120.06, 120.08, 125.43, 125.57,
3
127.32 (2C), 127.98 (2C), 141.31, 141.33, 143.39, 143.43, 151.56, 170.58, 172.92; Optical
22
rotation: [α] ꢀ 24.1° (c 0.332, CHCl ); IR (CHCl ): 3068 w, 3029 m, 2985 m, 2939 w, 2883
D
3
3
vw, 1797 s, 1758 s, sh, 1739 vs, 1724 vs, sh, 1609 vw, 1580 vw, 1479 w, 1463 m, 1452 s, 1421
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ꢀ1
w, 1386 s, 1377 m, 1305 vs, 1194 m, 1105 s, 1045 m, 1033 m, 621 w, 425 w cm ; ESI MS: 416
+
(
[M + Na] ); HR ESI MS: calcd for C H O NNa 416.14684; found 416.14694.
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Ethyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoate (8a).
A
solution of trimethylsilyl diazomethane (2M in diethyl ether, 7.9 mL, 15.81 mmol, 1.2 eq.) was
dissolved in dry THF (70 mL) under argon and cooled to ꢀ98 °C. A solution of
nꢀbutyllithium (2.5 M in hexanes, 6.5 mL, 16.21 mmol, 1.23 eq.) was added dropwise and the
solution was stirred at ꢀ98 °C for 30 min. The resultant mixture was transferred via cannula to a
solution of 7a (5.00 g, 13.18 mmol, 1 eq.) in dry THF (120 mL) at ꢀ116 °C. The reaction mixture
was stirred for 30 min at this temperature and then slowly warmed to ꢀ78 °C and quenched with
saturated NH Cl (150 mL). The aqueous phase was extracted with ethyl acetate (3×50 mL), the
4
combined organic layers were washed with brine (200 mL) and dried over anhydrous MgSO4.
The organic solvent was evaporated in vacuo. The residue was chromatographed on silica gel
1
(
CHCl /acetone 20:1) to afford the desired product 8a (4.42 g, 80%) as a yellowish solid. H
3
NMR (CDCl ): δ 1.29 (3H, t, J = 7.1), 1.93 – 2.10 (1H, m), 2.17 – 2.29 (1H, m), 2.33 – 2.53
3
(2H, m), 4.22 (3H, t, J = 7.1), 4.32 – 4.43 (3H, m), 5.27 (1H, bs), 5.56 (1H, d, J = 8.1), 7.32 (2H,
13
tt, J = 7.4, 1.3), 7.41 (2H, t, J = 7.5), 7.60 (2H, t, J = 6.6), 7.77 (2H, dd, J = 7.6, 1.0); C NMR
(
CDCl ): 14.30, 27.63, 36.56, 47.26, 53.63, 54.96, 61.89, 67.17, 120.11, 120.14, 125.22, 125.24,
3
1
27.20 (2C), 127.86 (2C), 141.40, 141.43, 143.78, 143.99, 156.18, 172.03, 193.65; Optical
22
rotation: [α] + 10.6° (c 0.265, CHCl ); IR (CHCl ): 3428 w, 3116 w, 3068 w, 2985 w, 2942
D
3
3
w, 2907 vw, 2110 s, 1740 s, sh, 1721 vs, 1642 m, 1510 s, 1478 w, 1465 w, 1451 m, 1381 s, 1105
ꢀ1
+
w, 1052 m, 1033 m, 622 w, 426 w cm ; ESI MS: 444 ([M + Na] ); HR ESI MS: calcd for
C H O N Na 444.15299; found 444.15292.
23
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Isopropyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-diazo-5-oxohexanoate (8b). A
solution of trimethylsilyl diazomethane (2M in diethyl ether, 6 mL, 11.93 mmol, 1.2 eq.) was
dissolved in dry THF (55 mL) under argon and cooled to ꢀ98 °C. A solution of nꢀbutyllithium
(2.5 M in hexanes, 4.9 mL, 12.23 mmol, 1.23 eq.) was added dropwise and the solution was
stirred at ꢀ98 °C for 30 min. The resultant mixture was transferred via cannula to a solution of
previous compound 7b (3.91 g, 9.94 mmol, 1 eq.) in dry THF (100 mL) at ꢀ116 °C. The reaction
10
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mixture was slowly warmed to ꢀ78 °C and then quenched with saturated NH Cl (100 mL). Then
4
the aqueous phase was extracted with ethyl acetate (3×50 mL), and the combined organic layers
were washed with water (40 mL), brine (40 mL) and dried over anhydrous MgSO . The organic
4
solvent was evaporated in vacuo. The residue was chromatographed on silica gel (CHCl /acetone
3
1
2
0:1) to afford the desired product 8b (3.68 g, 85%) as a yellowish solid. H NMR (CDCl ): δ
3
1.25 – 1.28 (6H, m), 1.95 – 2.04 (1H, m), 2.17 – 2.26 (1H, m), 2.31 – 2.52 (2H, m), 4.22 (1H, t, J
7.1), 4.29 – 4.43 (3H, m), 5.06 (1H, hept, J = 6.1), 5.27 (1H, s), 5.59 (1H, d, J = 8.2), 7.30 –
=
1
3
7
.34 (2H, m), 7.38 – 7.42 (2H, m), 7.59ꢀ 7.62 (2H, m), 7.75 – 7.77 (2H, m); C NMR (101
MHz, CDCl ): 21.81, 21.84, 27.69, 36.56, 47.21, 53.67, 67.10, 69.62, 120.08, 120.09, 125.18,
3
125.21, 127.16 (2C), 127.81 (2C), 141.35, 141.37, 143.75, 143.96, 156.16, 171.50, 193.67;
2
2
Optical rotation: [α] + 15.1° (c 0.674, CHCl ); IR (CHCl ): 3428 m, 3116 w, 3068 w, 2985
D
3
3
m, 2940 w, 2882 w, 2110 vs, 1731 vs, sh, 1719 vs, 1641 s, 1608 w, sh, 1580 vw, 1509 s, 1478 m,
466 m, 1451 s, 1418 w, sh, 1386 s, sh, 1377 s, 1349 s, 1232 s, 1105 s, 1052 s, 1033 m, 622 w,
1
ꢀ1
+
539 m, 488 m, 426 w cm ; ESI MS: 458 ([M + Na] ); HR ESI MS: calcd for C H O NaN
2
4
25
5
458.16873; found 458.16864.
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Ethyl 5-(diazomethyl)-3,4-dihydro-2H-pyrrole-2-carboxylate (9a) Compound 2a (117 mg,
0
.588 mmol, 1 eq.) was dissolved in CH CN (1 mL). The reaction mixture was heated for 2 h at
3
6
0 °C under inert atmosphere. The CH CN was evaporated and the crude product was purified
3
0
1
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by HPLC (CH CN/H O, HCOOH) affording a light orange oil (53 mg) in 50% yield. H NMR
3
2
(CDCl ): δ 1.27 (3H, t, J = 7.1), 2.83 – 2.93 (1H, m), 2.93 – 3.07 (2H, m), 3.09 – 3.20 (1H, m),
3
13
4
.24 (2H, dq, J = 7.1, 2.4), 5.17 (1H, dd, J = 9.2, 3.5), 7.43 (1H, s); C NMR (101 MHz,
CDCl ): 14.11, 19.98, 34.19, 59.06, 62.53, 126.82, 142.77, 168.70; IR (CHCl ): 2942 w, 2910 w,
3
3
2875 vw, 2103 vw, 1747 vs, 1475 w, 1676 m, 1605 vw, 1552 w, 1462 w, 1446 w, 1396 w, 1377
ꢀ1
+
m, 1202 vs, 1177 m, 1116 w, 1095 m, cm ; ESI MS: 182 ([M + H] ); HR ESI MS: calcd for
C H O N 182.0930; found 182.0931.
8
12
2
3
Isopropyl 5-(diazomethyl)-3,4-dihydro-2H-pyrrole-2-carboxylate (9b). Compound 2b (100
mg, 0.469 mmol, 1 eq.) was dissolved in CDCl (2 mL). The reaction mixture was stirred at rt
3
overnight. The CDCl was evaporated and the crude product was purified by HPLC
3
1
(
CH CN/H O, HCOOH) and a light orange oil (41 mg) was obtained in 45% yield. H NMR
3
2
(
CDCl ): δ 1.25 (3H, d, J = 6.1), 1.27 (3H, d, J = 6.1), 2.81 – 2.95 (2H, m), 2.96 – 3.06 (1H, m),
3
1
3
3
.08 – 3.18 (1H, m), 5.09 (1H, hept, J = 6.1), 5.13 (1H, dd, J = 9.1, 3.4), 7.43 (1H, s); C NMR
(101 MHz, CDCl ): 20.00, 21.69 (2C), 21.75, 34.24, 59.20, 70.48, 126.84, 142.71, 168.25;
3
22
Optical rotation: [α] ꢀ 31.8° (c 0.110, CHCl ); IR (CHCl ): 2104 w, 1741 vs, 1675 w, 1644
D
3
3
ꢀ1
w, 1551 w, 1465 m, 1388 m, sh, 1376 m, 1209 s, 1182 m, 1147 m, 1106 s, cm ; ESI MS: 196
+
([M + H] ); HR ESI MS: calcd for C H O N 196.10805; found 196.10808.
9
14
2
3
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,5-Dioxopyrrolidin-1-yl-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl carbonate (11). 4ꢀ
(hydroxymethyl)ꢀ5ꢀmethylꢀ1,3ꢀdioxolꢀ2ꢀone (10) (1.00 g, 7.89 mmol, 1 eq.) was dissolved in
dry Et O (30 ml) and the solution was cooled to 0 °C. At this temperature pyridine (608 mg, 619
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59
60
µL, 7.69 mmol, 1 eq.) was added followed by dropwise addition of a solution of Sꢀethyl
chlorothioformate (1.04 g, 874 µL, 8.38 mmol, 1.09 eq.) in dry Et O (8 mL). The mixture was
2
stirred for 1 h at 0 °C and overnight at rt (18 h). Et O was removed in vacuo and CH Cl (70 mL)
2
2
2
was added. The reaction mixture was washed with sat. NaHCO (40 mL) and with water (3×40
3
mL), dried with MgSO and CH Cl was removed by rotary evaporation. The crude product was
4
2
2
purified by column chromatography (hexane/EtOAc 5:1, R 0.24). A light yellow liquid (1.29 g)
f
was obtained in 77% yield. The intermediate Sꢀethyl Oꢀ((5ꢀmethylꢀ2ꢀoxoꢀ1,3ꢀdioxolꢀ4ꢀ
yl)methyl) carbonothioate (800 mg, 3.67 mmol, 1 eq.) and Nꢀhydroxysuccinimide (844 mg, 7.33
mmol, 2 eq.) were suspended in dry CH Cl (8 mL). The solution was cooled to 0 °C and
2
2
peracetic acid (836 mg (100 %), 2.32 g (36 %), 11.00 mmol, 3 eq., 36 % solution in acetic acid)
was added dropwise over 10 minutes. The final mixture was stirred for 30 minutes at 0 °C and 2
h at rt. CH Cl (20 mL) was added and the organic phase was washed with water (2×35 mL) and
2
2
sat. NaCl (35 mL). CH Cl was evaporated and the product was obtained as a colorless solid
2
2
1
compound (750 mg) in 76% yield. H NMR (CDCl ): δ 2.20 (3H, s), 2.86 (4H, s), 5.05 (2H, s);
3
1
3
C NMR (101 MHz, CDCl ): 9.67, 25.58 (2C), 59.89, 131.61, 142.20, 151.65, 168.39; IR
3
(
CHCl ): 1842 m, 1824 s, 1819 s, 1792 s, 1749 vs, 1431 w, 1386 w, 1309 m, 1195 s, 935 w, 900
3
ꢀ1
+
w, 811 vw cm ; ESI MS: 294 ([M + Na] ); HR ESI MS: calcd for C H O NNa 294.02204;
1
0
9
8
found 294.02213.
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Ethyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-6-diazo-5-
oxohexanoate (12a). FmocꢀNHꢀ(L)ꢀLeuꢀCOOH (874 mg, 2.47 mmol, 1.1 eq.) and HBTU (1023
mg, 2.70 mmol, 1.2 eq.) were suspended in dry DCM (15 mL). DIEA (872 mg, 1.18 mL, 6.75
mmol, 3 eq.) and then the solution of 2a (448 mg, 2.25 mmol, 1 eq.) in dry CH Cl (5 mL) were
0
1
2
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4
5
6
7
8
9
0
1
2
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6
7
8
9
0
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9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
added by syringe. The reaction mixture was stirred for 2 h at rt under inert atmosphere. CH Cl2
2
(
20 mL) was added and the organic phase was washed with sat. NaHCO (40 mL), 1M HCl (40
3
mL), water (2 x 40 mL) and sat. NaCl (40 mL), dried oved MgSO . The CH Cl was evaporated.
4
2
2
The crude product was purified by column chromatography (DCM/EtOAc 4:1, R 0.27) and light
f
1
yellow solid (949 mg) was obtained in 79% yield. H NMR (CDCl ): δ 0.95 (6H, d, J = 5.9),
3
1.26 (3H, t, J = 7.5), 1.50 – 1.59 (1H, m), 1.62 – 1.75 (2H, m), 1.95 – 2.05 (1H, m), 2.17 – 2.27
(1H, m), 2.29 – 2.45 (2H, m), 4.14 – 4.25 (4H, m), 4.34 – 4.45 (2H, m), 4.49 – 4.58 (1H, m),
5.19 (1H, s), 5.27 (1H, d, J = 8.4), 6.80 (1H, d, J = 7.6), 7.31 (2H, t, J = 7.4), 7.40 (2H, t, J =
13
7.8), 7.59 (2H, d, J = 7.5), 7.76 (2H, d, J = 7.6); C NMR (CDCl ): 14.12, 22.03, 22.92, 24.66,
3
2
6.96, 36.33, 41.65, 47.14, 51.94, 53.56, 55.00, 61.73, 67.09, 120.00, 120.03, 125.05, 125.13,
27.12 (2C), 127.76, 127.77, 141.29, 143.64, 143.75, 143.79, 156.15, 171.46, 172.26, 193.81;
1
2
2
Optical rotation: [α] ꢀ 9.7° (c 0.109, CHCl ); IR (CHCl ): 3428 m, 3326 w, 3116 vw, 3068
D
3
3
w, 2110 s, 1793 w, 1732 vs, 1720 vs, sh, 1678 vs, 1642 m, 1509 vs, 1478 m, 1451 s, 1376 s,
ꢀ1
+
1
032 m, 622 w, 426 w, cm ; ESI MS: 557 ([M + Na] ); HR ESI MS: calcd for C H O N Na
29 34 6 4
557.23706; found 557.23707.
Isopropyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methylpentanamido)-6-
diazo-5-oxohexanoate (12b). FmocꢀNHꢀ(L)ꢀLeuꢀCOOH (1.8 g, 5.16 mmol, 1.1 eq.) and TBTU
1.81 g, 5.63 mmol, 1.2 eq.) were suspended in CH Cl (40 mL) and diisopropylethyl amine
(
2
2
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(
2.51 mL, 14.07 mmol, 3 eq.) was added. The reaction mixture was stirred at room temperature
for 30 min and then a solution of 2b (1.00 g, 4.69 mmol, 1 eq.) in dry CH Cl (20 mL) was added
2
2
by syringe. The reaction mixture was stirred for 2 h at room temperature under inert atmosphere.
The organic solvent was evaporated in vacuo. The residue was chromatographed on silica gel
10
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55
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(hexane/EtOAc 3:2 to 1:1) to afford the desired product (1.85 g, 72%) as a yellow amorphous
1
solid. H NMR (CDCl ): δ 0.91 – 0.96 (6H, m), 1.23 – 1.25 (6H, m), 1.51 – 1.73 (3H, m), 1.93 –
3
2
.02 (1H, m), 2.14 – 2.24 (1H, m), 2.26 – 2.44 (2H, m), 4.16 – 4.24 (2H, m), 4.33 – 4.37 (1H,
m), 4.39 – 4.44 (1H, m), 4.47 – 4.52 (1H, m), 4.96 – 5.08 (1H, m), 5.18 (1H, s), 5.36 (1H, d, J =
.2), 6.84 (1H, d, J = 7.8), 7.30 (2H, tt, J = 7.4, 1.2), 7.37 – 7.42 (2H, m), 7.58 (2H, d, J = 7.4),
8
1
3
7.75 (2H, d, J = 7.5); C NMR (CDCl ): 21.70, 21.72, 22.04, 22.92, 24.66, 27.04, 36.37, 41.75,
3
47.14, 52.05, 53.56, 54.85, 67.08, 69.47, 119.98, 120.01, 125.06, 125.13, 127.10, 127.11, 127.74,
22
127.75, 141.28 (2C), 143.76, 143.81, 156.14, 170.95, 172.29, 193.80; Optical rotation: [α]
ꢀ
D
6.1° (c 0.472, CHCl ); IR (CHCl ): 3304 m, sh, 3067 w, 3018 w, 2105s, 1730 s, 1704 s, 1659 vs,
3
3
1639 sh, m, 1612 sh, w, 1580 sh, w, 1539 s, 1478 m, 1451 m, 1467 m, 1386 sh, s, 1375 s, 1244 s,
ꢀ1
1
172 sh, m, 1145 m, 1106 s, 834 sh, w,759 m, 740 s, 621 m, 427 cm ; ESI MS: 571 ([M +
+
Na] ); HR ESI MS: calcd for C H O N Na 571.25271; found 571.25271.
2
4
33
4
4
Ethyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1H-indol-3-yl)propanamido)-6-
diazo-5-oxohexanoate (12c). FmocꢀNHꢀ(L)ꢀTrpꢀCOOH (353 mg, 0.828 mmol, 1.1 eq.) and
HBTU (343 mg, 0.904 mmol, 1.2 eq.) were suspended in dry CH Cl (5 mL). DIEA (292 mg,
2
2
394 µL, 2.26 mmol, 3 eq.) was added followed by a solution of 2b (150 mg, 0.753 mmol, 1 eq.)
in dry CH Cl (3 mL) were added by syringe. The reaction mixture was stirred for 2 h at rt under
2
2
inert atmosphere. CH Cl (20 mL) was added and the organic phase was washed with sat.
2
2
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NaHCO (20 mL), 1M HCl (20 mL), water (30 mL) and sat. NaCl (30 mL), dried over MgSO .
3
4
DCM was evaporated. The crude product was purified by LC (CH Cl /EtOAc 2:1, R 0.21) and
2
2
f
1
light yellow solid (303 mg) was obtained in 66% yield. H NMR (CDCl ): δ 1.22 (3H, t, J =
3
0
1
2
3
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8
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2
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7.2), 1.82 – 1.93 (1H, m), 2.00 – 2.26 (3H, m), 3.18 (1H, dd, J = 14.5, 7.1), 3.38 (1H, dd, J =
14.0, 5.3), 4.09 (2H, q, J = 8.2), 4.20 (1H, t, J = 7.1), 4.32 – 4.48 (3H, m), 4.50 – 4.59 (1H, m),
5
.05 (1H, bs), 5.51 (1H, d, J = 7.9), 6.61 (1H, d, J = 7.5), 7.07 (1H, bs), 7.13 (1H, t, J = 7.4),
.20 (1H, t, J = 7.2), 7.30 (2H, t, J = 7.5), 7.35 (1H, d, J = 8.0), 7.40 (2H, t, J = 7.5), 7.57 (2H, t,
7
1
3
J = 6.6), 7.67 (1H, d, J = 7.9), 7.76 (2H, d, J = 7.6), 8.28 (1H, bs); C NMR (101 MHz, CDCl ):
3
14.20, 27.09, 28.51, 36.20, 47.25, 52.13, 54.86, 55.76, 61.77, 67.25, 110.29, 111.39, 118.88,
120.00, 120.10, 120.11, 122.41, 123.66, 125.25, 125.29, 126.92, 127.22 (2C), 127.62, 127.86
2
2
(2C), 136.38, 141.40, 143.87, 143.96, 156.09, 171.28, 171.48, 193.84; Optical rotation: [α]
ꢀ
D
28.6° (c 0.178, DMF); IR (KBr): 3424 s, 3308 m, sh, 2978 w, 2106 m, 1728 s, 1697 m, sh, 1654
m, 1519 m, 1478 w, 1450 m, 1382 m, sh, 1376 m, 1343 m, 1289 m, 1224 m, 1104 mꢀw, 1081 mꢀ
ꢀ1
w, 1040 m, 1032 m, 877 w, 855 w, 760 m, 742 m, 621 m, 427 w, cm ; ESI MS: 630 ([M +
+
Na] ); HR ESI MS: calcd for C H O N Na 630.23230; found 630.23236.
3
4
33
6
5
Isopropyl-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1H-indol-3-
yl)propanamido)-6-diazo-5-oxohexanoate (12d). FmocꢀNHꢀ(L)ꢀTrpꢀCOOH (880 mg, 2.06
mmol, 1.1 eq.) and HBTU (854 mg, 2.25 mmol, 1.2 eq.) were suspended in dry DMF (14 mL).
DIEA (727 mg, 980 µL, 5.63 mmol, 3 eq.) was added followed by a solution of 2b (400 mg, 1.88
mmol, 1 eq.) in dry DMF (5 mL). The reaction mixture was stirred for 4 h at rt under inert
atmosphere. DMF was evaporated, DCM (100 mL) was added and the organic phase was washed
with sat. NaHCO (100 mL), water (100 mL), 1M HCl (100 mL), water (100 mL) and sat. NaCl
3
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(
100 mL), dried oved MgSO . DCM was evaporated. The crude product was purified by column
4
chromatography (DCM/EtOAc 5:1, R 0.15) and light yellow solid (540 mg) was obtained in
f
1
4
6% yield. H NMR (CDCl ): δ 1.19 (3H, d, J = 6.3), 1.23 (3H, t, J = 6.3), 1.81 – 1.92 (1H, m),
3
10
11
12
13
14
15
16
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19
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2
.01 – 2.28 (3H, m), 3.18 (1H, dd, J = 14.5, 7.1), 3.39 (1H, dd, J = 14.2, 5.2), 4.20 (1H, t, J =
7.1), 4.30 – 4.48 (3H, m), 4.54 (1H, q, J = 6.9), 4.88– 4.99 (1H, m), 5.07 (1H, bs), 5.50 (1H, d, J
7.9), 6.59 (1H, d, J = 7.4), 7.07 (1H, bs), 7.14 (1H, t, J = 7.0), 7.20 (1H, t, J = 7.2), 7.30 (2H,
tdd, J = 7.5, 2.5, 1.1), 7.36 (1H, d, J = 8.0), 7.40 (2H, t, J = 7.5), 7.56 (2H, t, J = 6.6), 7.67 (1H,
=
1
3
d, J = 7.9), 7.77 (2H, d, J = 7.6), 8.23 (1H, bs); C NMR (101 MHz, CDCl ): 21.78, 21.83,
3
2
7.20, 28.47, 36.20, 47.23, 52.19, 54.91, 55.75, 67.23, 69.22, 110.28, 111.40, 118.88, 120.03,
120.10, 120.12 (2C), 122.45, 123.66, 125.25, 125.30, 127.23 (2C), 127.61, 127.86 (2C), 136.35,
22
1
41.40 (2C), 143.85, 143.96, 156.09, 170.80, 171.42, 193.92; Optical rotation: [α] ꢀ 32.0° (c
D
0
.193, DMF); IR (KBr): 3424 s, 3300 m, 3130 vw, 2980 w, 2932 s, 2110 m, 1722 s, 1695 s,
1654 s, 1625 m, 1547 m, sh, 1532 m, 1520 m, sh, 1478 w, 1385 m, 1375 m, 1353 m, 1343 m,
1
288 m, 1236 m, 1182 w, 1145 m, 1105 m, 1031 mꢀw, 1010 w, 852 w, 798 w, 758 m, 741 m,
ꢀ1
+
6
21 w, 427 wꢀm, cm ; ESI MS: 644 ([M + Na] ); HR ESI MS: calcd for C H O N Na
35 35 6 5
644.24795; found 644.24811.
Mice Efficacy Studies
All mouse efficacy studies were conducted according to protocol #MO13M69 approved by the
Animal Care and Use Committee at Johns Hopkins University. Female athymic (RHꢀFoxn1nu
mice) mice between 25 and 30g were obtained (Harlan Sprague Dawley Inc, Indianapolis,
Indiana), and maintained on a 12 hour lightꢀdark cycle with ad libitum access to food and water.
U87 human glioma cells were obtained as a gift from the lab of Dr. Gregory Riggins (Johns
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