One-pot fluorescent labeling protocol for complex hydroxylated bioactive natural products

Article abstract of DOI:10.1021/jo401559t

Tagging of small bioactive molecules with a fluorophore is a highly sensitive method to trace their cellular activities through real-time visual information. Here we disclose a 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-based, high-yielding, one-pot labeling protocol for hydroxylated molecules using Yamaguchi coupling as the key reaction. This methodology was successfully applied on several sensitive and complex hydroxylated bioactive compounds including 7-deacetylazadiradione, simvastatin, camptothecin, andrographolide, cinchonine, β-dihydroartemisinin, and azadirachtin A. Further, utility of this protocol was illustrated on the cytotoxic activity of azadiradione derivatives against several cancer cell lines through cell imaging of two qualified fluorescent probes.

Full text of DOI:10.1021/jo401559t

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Article
One-Pot Fluorescent Labeling Protocol for
Complex Hydroxylated Bioactive Natural Products
Saikat Haldar, Santosh Kumar, Swati P Kolet, Harshal S. Patil,
Dhiraj Kumar, Gopal C Kundu, and Hirekodathakallu V Thulasiram
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo401559t • Publication Date (Web): 30 Sep 2013
Downloaded from http://pubs.acs.org on October 6, 2013
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One-Pot Fluorescent Labeling Protocol for Complex
Hydroxylated Bioactive Natural Products
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†
‡, #
†, #
†
‡
Saikat Haldar, Santosh Kumar, Swati P. Kolet, Harshal S. Patil, Dhiraj Kumar, Gopal
‡
*, †, §
C. Kundu, Hirekodathakallu V. Thulasiram
†
Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory,
Pune-411008, India.
§
CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi-110007, India.
‡
National Center for Cell Science, Pune-411007, India.
Corresponding Author. Fax: +91 2025902636; Tel: +91 2025902478; E-mail:
hv.thulasiram@ncl.res.in
#
Author Contributions. These authors contributed equally.
Table of Contents (TOC) Graphic.
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ABSTRACT
Tagging of small bioactive molecules with fluorophore is a highly sensitive method to trace their
cellular activities through real-time visual information. Here we disclose a 7-nitrobenzo-2-oxa-
1,3-diazole (NBD) based high yielding, one-pot labeling protocol for hydroxylated molecules
using Yamaguchi coupling as the key reaction. This methodology was successfully applied on
several sensitive and complex hydroxylated bioactive compounds including 7-
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deacetylazadiradione,
simvastatin,
camptothecin,
andrographolide,
cinchonine,
β-
dihydroartemisinin and azadirachtin A. Further, utility of this protocol was illustrated on the
cytotoxic activity of azadiradione derivatives against several cancer cell lines through cell
imaging of two qualified fluorescent probes.
INTRODUCTION
Fluorescence is a highly sensitive technique for the real time monitoring of cellular phenomena
through qualitative and quantitative optical information obtained from the fluorescence detecting
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instruments. Natural products or biochemical probes covalently linked with the fluorescent
organic dyes are used to unveil the cellular processes (including localization or specific
interactions) involved with their activity. Fluorescence imaging of the living cells to study the
cellular events or visualize the associated phenotypes has become an attractive technique in
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recent years. Therefore, exploration of rapid, inexpensive and effective fluorescent tagging
protocol for the bioactive natural products and successful implementation of the developed
procedure will facilitate the integration of fluorescence imaging and investigation of cellular
events.
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Reported protocols for the fluorescent labeling of natural products involving several reaction
steps along with purification and protection-deprotection procedures result in low overall yield
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and make the process tedious.
Indeed the availability of natural products is scanty in most of
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the cases due to their low abundance in the natural sources, making it difficult to carry out all the
required steps. Secondly, the widely used tags (such as fluorescein, rhodamine) are bulkier in
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nature, which may lead to loss of biological activity. Although, ready to use fluorescent tags
are available commercially, they are quite expensive and require extreme reaction conditions for
labeling (e.g. NBD-F or DBD-COCl needs 60 ºC at basic pH) which restrict their use for tagging
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the sensitive multifunctional natural products.
Consequently, there is a need for the
development of an inexpensive, mild and high-yielding protocol for the fluorescent labeling of
biologically active complex natural products capable of maintaining the activity in the labeled
derivative. In this article we have demonstrated a one-pot labeling protocol for the hydroxylated
bioactive compounds by a 7-nitrobenzo-2-oxa-1,3-diazole (NBD) based tag which was
successfully applied to label several complex multifunctional molecules. Further, two semi-
synthetic derivatives of azadiradione (as azadiradione and its derivatives are known for their
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cytotoxic activity against several human cancer cell lines)
and their fluorescently labeled
analogues were subjected to comparative cytotoxicity studies. Also, the cellular uptake and
intracellular localization of these labeled compounds in various cancer cell lines were monitored
by fluorescence imaging.
RESULTS AND DISCUSSION
Chemistry. NBD-Cl is a non-fluorescent molecule, which undergoes nucleophilic substitution
by amine functionality and becomes a highly sensitive fluorescent entity (depending on the
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polarity of the environment) (Supporting information). This technique is being used over the
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years for the labeling of proteins and amine containing small molecules.
But, the reactivity
of NBD-Cl towards hydroxyl group is very poor and this method can’t be used directly for the
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tagging of hydroxylated molecules. Here, our protocol involves the aromatic nucleophilic
substitution on NBD-Cl by the amine functionality of amino acids in the initial step to generate
fluorescent reagent followed by coupling between the free carboxylic end and the hydroxylated
bioactive molecule in the same vessel (Scheme 1). It is always preferable to synthesize
fluorescent reagent freshly in one-pot rather than using it as an individual tagging dye due to
several reasons: (i) length of the amino acid used in the initial step determines the length of
spacer between the tag and natural product. Therefore, for achieving different spacer lengths in
the labeled analogues, individual fluorescent reagents of various spacer lengths have to be
prepared and purified separately. Instead, it is more convenient to use one-pot protocol utilizing
the commercially available amino acids of own choice corresponding to the desired spacer
length. (ii) Fluorescent reagents are photosensitive and they require special care during
preparation and storage such as dim light and low temperature. Preferably they are used
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immediately after preparation and purification. Therefore, it is more convenient to generate
fluorescent reagent freshly in one-pot from two non-fluorescent starting materials (NBD-Cl and
amino acids). (iii) After the preparation of fluorescent reagent it is essentially need to be purified
before storage. But, the reported one-pot protocol is devoid of one additional purification step
making it less tedious.
Scheme 1. One-pot protocol for the fluorescent labeling of hydroxylated bioactive molecules.
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Comparative Kinetics among Coupling Reactions. Among several well-established
coupling reactions available between carboxylic acid and alcohol, Yamaguchi was preferred on
the basis of outcome of comparative kinetic experiment. 1 was used as the model substrate and
allowed to couple with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid in presence
of various coupling reagents (DCC, EDC, DIC, Yamaguchi) (Figure 1). Progress of the reactions
was monitored by HPLC using the standard graphs prepared for 1 and 1a. The coupling reagents,
DCC, EDC and DIC showed similar reaction kinetic profile. About 50-60% of 1 (by mole) was
converted to 1a in 90 min of reaction time by all three coupling agents. However, no significant
change in the product level was observed till 10 h (Figure 1). The higher stoichiometric ratios of
coupling reagents (DCC, EDC and DIC) also didn’t alter the progress or rate of the reaction
significantly even after the prolonged reaction time (Supporting information). On the other hand,
Yamaguchi coupling showed quantitative conversion in 30 min; hence was chosen as the
preferred coupling reaction for other molecules.
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Figure 1. Comparative kinetics among various well-known coupling reactions between 1 and 6-
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N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid.
Flexibility in Spacer Length. The choice of amino acid to be used in the initial step will
determine the length of spacer joining the NBD skeleton and the bioactive molecule of interest.
Indeed, the length of the spacer can vary the hydrophobicity of the labeled molecule which might
be a guiding factor for the cellular uptake. Also, spacer of optimum length is important for
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retaining the activity observed with parental molecule. This flexibility in our methodology
makes it unique and will be helpful for getting a successful labeling on a complex molecule with
preserved activity. To demonstrate, 1 was labeled with spacers of three different lengths using
three unlike amino acids; 6-aminocaproic acid (six carbon spacer), 11-aminoundecanoic acid
(eleven carbon spacer) and L-proline (creating turn in the structure) (Scheme 2).
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Scheme 2. Selection of the amino acid in the initial step varies the length of spacer in between
the tag and studied molecule.
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Reagents and conditions: (i) NBD-Cl, NaHCO , acetonitrile, water, 55 ºC; (ii) 1, 2,4,6-
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trichlorobenzoylchloride, TEA, DMAP, anhydrous THF, 85-90% for two steps.
Nature of the Hydroxyl Group. To investigate the efficiency of Yamaguchi coupling with
varying nature of hydroxyl groups, several structurally simple compounds bearing different kind
of hydroxyl functionalities (such as phenolic, primary, secondary and tertiary) were chosen and
subjected to one-pot protocol using 6-aminocaproic acid (Scheme 3). Eugenol (2, phenolic
hydroxyl), (-)-borneol (3, secondary hydroxyl), α-santalol (4, allylic primary hydroxyl), (-)-
dihydrocarveol (5, secondary hydroxyl) and one partially protected mannose (6) carrying
primary hydroxyl furnished the labeled product with excellent yields. However, the coupling did
not work out when α-(-)-bisabolol, (7) a sesquiterpene carrying tertiary hydroxyl group was used
as the substrate. These results indicated that the developed protocol for NBD based fluorescent
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tagging of bioactive molecules possessing phenolic, primary and secondary hydroxyl groups
using Yamaguchi coupling was highly efficient.
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Scheme 3. Efficiency of Yamaguchi coupling varies with the nature of hydroxyl group.
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Applicability with Complex Molecules. To showcase broad applicability of the present
methodology several structurally diverse and multifunctional molecules were selected for
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fluorescent labeling (Table 1) using above discussed one-pot protocol. The fluorescent tagged
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compound (8a) was obtained in good yield (71%), when nimbocinol (8, deacetylated
azadiradione) carrying a sterically hindered axial hydroxyl group at C-7 of basic limonoid
skeleton was used as the substrate. Similarly, cholesterol (9), 11α-hydroxyprogesterone (10) and
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diosgenin (11, a steroidal sapogenin containing a spiroketal moiety) furnished corresponding
tagged compounds 9a, 10a and 11a in good to excellent yields. Phenolic hydroxyl on the
isoindolin skeleton of compound 12 responded to the reaction with an excellent yield. Ezetimibe
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(13, a cholesterol lowering drug) possessing both alkylic and phenolic hydroxyl underwent a
competitive reaction. However, at -15 ºC with 5 min of reaction time, tagged phenolic hydroxyl
was obtained as a major product (52%) (For the detailed characterization consult supporting
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information) along with doubly tagged product (27%). An antimalarial alkaloid cinchonine (14)
carrying a secondary hydroxyl group was labeled with very good yield (82%) whereas another
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anticancer alkaloid (S)-(+)-camptothecin (15) yielded the coupled product with a poor yield
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09%) due to the tertiary nature of its hydroxyl functionality. A hypolipidemic drug simvastatin
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derivative with 81% yield. Andrographolide (17), an anticancer natural product has three
different hydroxyl groups around its skeleton; an allylic secondary hydroxyl on five membered
lactone ring, one primary and another hindered secondary hydroxyl group. Although at room
temperature the same reaction condition produced a mixture of products, at -15 ºC with 5 min
stirring the mono-labeled (on allylic secondary hydroxyl) product was obtained with a moderate
yield (33%) and characterized by 1D- and 2D-NMR studies as discussed in the supporting
information. Interestingly, a reduced product of salanin (18) having
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Table 1. Entries showing applicability of the developed one-pot protocol for fluorescent
labeling of complex bioactive hydroxylated compounds.
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Entry
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No reaction
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trichlorobenzoylchloride, TEA, DMAP, anhydrous THF. Parentheses are showing numbering of
compounds.
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two hindered secondary hydroxyl and another primary hydroxyl groups, furnished mono-labeled
product on primary hydroxyl with an excellent yield (93%) (For the detailed characterization
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consult supporting information). β-dihydroartemisinin (19) a well-known antimalarial agent
having a sensitive peroxo bridge along with an adjacent ketal moiety in its skeleton was also able
to produce the coupled product with 71% yield. A well-studied highly potent antifeedant neem
limonoid azadirachtin A (20) possesses a unique molecular architecture carrying ester, epoxide,
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ether and ketal functionalities which makes it highly sensitive towards pH and light.
Surprisingly, it was not able to furnish any product and remained unreacted under the same
reaction condition despite having three hydroxyl groups around its skeleton. Inability of
azadirachtin A (20) to produce any product may be due to the extreme steric hindrance around
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hydroxyl groups and their involvement in the intramolecular hydrogen bonding network. To
avoid these obstacles, an alternative approach was adopted.
Scheme 4. Fluorescent labeling protocol for azadirachtin A.
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Initially, a short spacer carrying a protected hydroxyl group at the tail was attached followed by
deprotection and Yamaguchi coupling. Azadirachtin A (20) was reacted with a propargyl
bromide derivative carrying a TBDMS protected hydroxyl group at the other end in presence of
sodium hydride. After nucleophilic substitution by 11-OH (as characterized on the basis of
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earlier reports),
hydroxyl was deprotected by TBAF and further subjected to one-pot protocol
to get labeled azadirachtin (20c) in 24% overall yield (Scheme 4).
Biology. To assess the applicability of fluorescent tagged bioactive molecules synthesized in
this study (Scheme 2, Table 1), two limonoids were chosen for their biological evaluation and
cellular imaging. Two azadiradione derivatives (1 and 8) and their corresponding labeled
analogues (1a and 8a) were evaluated for cytotoxic activity against several cancer cell lines and
further they were used for cell imaging studies.
Cancer Cell Viability Studies. MDA-MB-231 cells were treated with either of labeled (1a
and 8a) and parent (1 and 8) azadiradione derivatives with varying concentrations (0-100 µg/mL)
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and MTT assay was performed. The results depicted comparable decrease in cell viability in
labeled and unlabeled limonoids in a concentration dependent manner (Figure 2).
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Figure 2. (A &B) MDA-MB-231 cells were treated with labeled and unlabeled limonoids and
cell viability was assessed by MTT assay. The data obtained were analyzed statistically and
represented graphically. Column, mean + SE, *P<0.001, **P=0.022, #P=0.1, ##P=0.074 vs.
control. The results are the representative of three independent experiments.
Cancer Cell Motility Assay. Cancer metastasis is basically the migration of tumor cells from
the primary tumors to the distant body parts to develop secondary tumors. Any potent drug
should target cancer cell viability as well as motility. Therefore to check the effect of labeled and
unlabeled limonoids on breast cancer cell motility, wound migration assay was performed.
Confluent monolayer of MDA-MB-231 cells were wounded uniformly, treated with labeled (1a
and 8a) as well as unlabeled (1 and 8) limonoids (0-20 µg/mL) for 12 h and motility was
assessed. The results demonstrated the inhibition in MDA-MB-231 cell motility with increasing
concentration of limonoids (labeled as well as unlabeled) (Figure 3).
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Figure 3. Wound migration is inhibited by limonoids in MDA-MB-231 cells. Cells were treated
with 1, 1a, 8 and 8a (0-20 µg/mL) and wounds were photographed after 12 h. Graphical
representation for the quantification of wound migration assay; Column, mean + SE, *P<0.002,
#
P=0.01 vs. control.
Cell Imaging Studies. The comparative inhibition studies of cancer cell viability and motility
have indicated the concentration dependent cytotoxic activity for both the labeled analogues (1a
or 8a) and comparable inhibitory potencies with respect to the parent azadiradione derivatives.
The retention of activity in labeled analogues may be attributed to the smaller size of NBD and
presence of highly flexible six-carbon spacer joining the tag and bioactive molecule. However,
the slight alternation in potency of tagged derivatives with respect to the parent molecules may
be due to the structural modification caused by the attachment of NBD tag and esterification of
hydroxyl functionality. Therefore, considering the extent of cytotoxic activity preserved, 1a and
8
a can be considered as the valid cancer cell imaging probe for azadiradione derivatives.
Cancer cells (A375, HeLa and MDA-MB-231) were treated either with 1a or 8a (20 μg/mL)
for 1 h and analyzed for cellular internalization and localization under confocal microscope.
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Results depicted the cellular uptake of NBD labeled compounds irrespective of the cancer cell
lines and no specific localization in the nucleus (Figure 4). Further these imaging probes can be
used for studying the localization in sub-cellular organelles. To demonstrate specific examples,
HeLa cells treated either with 1a or 8a were stained by Mito-tracker Red and analyzed through
confocal microscopy, which didn’t show any colocalization with mitochondria (Supporting
information). Similarly, the colocalization experiment was performed with ER-tracker red and
labeled analogues (1a and 8a) were found to be localized in the endoplasmic reticulum (ER) on
the basis of yellow fluorescence observed in the overlaid image (Figure 5). Incubation of 1a with
A375 cells and analyses of the cell lysate after the incubation period indicated that the ester bond
between tag and compound 1 is stable under intracellular conditions (Supporting information).
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Figure 4. Cancer cells (A375, Hela and MDA-MB-231) were treated (20 µg/mL) with NBD
labeled limonoids (1a and 8a) for 1 h and analyzed under confocal microscope. Green:
compounds labeled with NBD, Blue: Nucleus staining with DAPI. Micron bar: 50 µm.
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Figure 5. (A) MDA-MB-231 cells were co-stained with 1a (20 µg/mL) and ER-tracker red (1
μM) and analyzed under confocal microscopy. (B) MDA-MB-231 cells were co-stained with 8a
(20 µg/mL) and ER-tracker red (1 μM) and analyzed under confocal microscopy. Nuclei were
stained with DAPI. Blue: Nucleus; Red: ER; Green: NBD-labeled limonoids. Micron bar: 10
µm.
CONCLUSIONS
Simple and highly efficient one-pot methodology for the NBD based fluorescent labeling of
hydroxylated small molecules was developed, which can be applied to track their mode of action
through fluorescence cell imaging. Among several common coupling reactions, Yamaguchi was
found to be best on the basis of yield and kinetic studies. Protocol was executed with a mild
condition so that it was able to sustain sensitive functionalities such as lactone, ketal, ether,
peroxo or epoxide in the reaction environment. Both nucleophilic substitution and coupling steps
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were carried out in the same vessel and required single purification step. Even the natural
products isolated in trace amount (a few milligrams) can be labeled in sufficient yield and more
economic way using the present protocol. Importantly, coupling reaction was very selective
between acid and alcohol functionalities, which allows to tag the complex molecules with
multiple functional groups. Fluorescein, a widely used fluorescent tag, was replaced by NBD as
it is less bulkier than former. Again, length of the spacer can easily be modified by a suitable
choice of the amino acid. Further, evaluation of the two NBD tagged azadiradione derivatives for
cytotoxic activity indicated that the labeling did not alter the activity drastically. The
internalization and localization of those probes were imaged successfully inside the cancer cells
and found to be localized in the endoplasmic reticulum. Therefore, this methodology can be used
to synthesize the fluorescent imaging probes of hydroxylated bioactive compounds to study the
cellular processes especially cellular uptake, localization or specific interactions.
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EXPERIMENTAL SECTION
General procedure: (A) One-pot labeling protocol. A solution of NBD-Cl (1.00 equiv) in
acetonitrile (18 mL/mmol) was added dropwise to a solution of amino acid (1.00 equiv) and
sodium bicarbonate (3.00 equiv) in water (6 mL/mmol) at 55 ºC and incubated for 1 h. Then
acetonitrile was concentrated under reduced pressure and pH of the aqueous reaction mixture
was adjusted to ~2.0 using 1N HCl. Further it was concentrated to dryness under 20 mbar
pressure and 62 ºC temperature. The deep orange crude solid was again dissolved in minimum
amount of acetonitrile and dried in the same condition to make sure that there is no residual
moisture remaining. Further Yamaguchi coupling was carried out in the same vessel following
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the reported procedure with slight modifications. The natural product/bioactive molecule (0.75
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equiv) with hydroxyl functionality in anhydrous THF (20 mL/mmol) was added to the dried
crude under inert atmosphere with stirring. 2,4,6-trichlorobenzoyl chloride (1.00 equiv) and
anhydrous TEA (1.00 equiv) were successively added dropwise to the reaction mixture. After 5
min, DMAP (1.00 equiv) was added to the reaction vessel and continued with stirring for another
30 min. The reaction was quenched by adding few drops of water. Then it was concentrated to
dryness and directly purified over silica gel column. All the steps were performed under dim
light and labeled compounds were stored at -20 ºC in dark.
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(B) Coupling reaction (DCC, EDC and DIC). To a mixture of acid (1.00 equiv), alcohol
(0.75 equiv) and DMAP (1.00 equiv) in an inert atmosphere, anhydrous DCM (30 mL/mmol)
was added. The mixture was stirred to dissolve all the components followed by addition of
coupling reagent (1.00 equiv) (DCC, EDC or DIC) in stirring condition. After addition, reaction
mixture was left for stirring at room temperature.
Preparation of 21a. Azadirachtin A (100 mg, 0.14 mmol) taken in argon atmosphere was
dissolved in 1.0 mL of anhydrous DMF and the mixture was cooled to 0 ºC. 60% sodium hydride
(10.2 mg, 0.42 mmol) was added to the mixture and stirred at 0 ºC for 8 min. The colourless
reaction mixture became light brown by this time period. Then propargyl bromide derivative was
added (52.6 mg, 0.20 mmol) to it followed by a catalytic amount (pinch) of TBAI. Continuing
the stirring at 0 ºC for another 4 min, the reaction mixture was shifted to room temperature and
allowed to stir for 20 min. After quenching the reaction with few drops of chilled water, it was
taken in ethyl acetate (20 mL) and washed thrice with brine solution (50 mL). Ethyl acetate layer
was concentrated and purified by silica gel column chromatography using gradient mixture of
DCM and methanol as eluent to get the desired product (21a) (46.72 mg, 0.052 mmol, yield 37
%
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Preparation of 21b. 1M TBAF (62 μL, 0.062 mmol) was added dropwise to 21a (46.72 mg,
0
.052 mmol) taken in 2 mL of anhydrous THF and the reaction mixture was stirred for 10 min in
an inert atmosphere. Further it was concentrated and purified over silica gel column using 2.0%
methanol in DCM as eluent to produce the deprotected product 21b in 92% yield (37.7 mg,
0.048 mmol).
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Analytical data. (3S,7R,8R,10S,13S,17R)-7-acetoxy-17-(furan-3-yl)-4,4,8,10,13-pentamethyl-
1
6-oxo-2,3,4,5,6,7,8,9,10,11,12,13,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 6-
((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (1a): 41.9 mg (0.057 mmol) of 1a was
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isolated from 30.0 mg (0.066 mmol) of 1 with 87% yield. Orange solid. [α] D = -15.5 (c 3.0,
-
1
1
CHCl ). IR (CHCl ) ν (cm ): 3327, 1719, 1700. H NMR (CDCl , 400 MHz) δ: 8.47 (d,
3
3
max
3
J=8.55 Hz, 1H), 7.45 (m, 1H), 7.41 (m, 1H), 6.52 (m, 1H), 6.25 (m, 1H), 6.17 (d, J=8.54 Hz,
H), 5.83 (s, 1H), 5.25 (m, 1H), 4.53 (m, 1H), 3.53 (dd, J=12.51, 6.41 Hz, 2H), 3.37 (s, 1H),
1
1
3
2.37 (m, 2H), 1.95 (s, 3H), 1.25 (s, 3H), 1.02 (s, 3H), 0.97 (s, 3H), 0.86 (s, 3H), 0.77 (s, 3H). C
NMR (CDCl , 100 MHz) δ: 205.5, 193.5, 173.1, 169.7, 144.2, 143.9, 142.6, 141.5, 136.5, 123.8,
3
1
23.1, 118.6, 111.2, 98.5, 80.2, 74.7, 60.5, 48.1, 48.0, 44.0, 43.6, 43.1, 37.3, 37.2, 34.2, 30.3,
+
28.0, 27.6, 26.3, 26.2, 26.1, 24.3, 23.3, 22.8, 21.0, 16.4, 15.7, 15.5. HRMS (ESI) m/z: [M+H]
Calcd for C H N O , 731.3659; found, 731.3656. Purity (HPLC): 97.8%.
4
0
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(
3S,7R,8R,10S,13S,17R)-7-acetoxy-17-(furan-3-yl)-4,4,8,10,13-pentamethyl-16-oxo-
2,3,4,5,6,7,8,9,10,11,12,13,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)undecanoate (1b): 47.3 mg (0.059 mmol) of 1b was
11-((7-
2
5
isolated from 30.0 mg (0.066 mmol) of 1 with 89% yield. Orange solid. [α] = -12.6 (c 1.7,
D
-
1
1
CHCl ). IR (CHCl ) ν (cm ): 3327, 1715, 1700. H NMR (CDCl , 400 MHz) δ: 8.50 (d,
3
3
max
3
J=8.72 Hz, 1H), 7.46 (m, 1H), 7.42 (m, 1H), 6.35 (m, 1H), 6.27 (m, 1H), 6.17 (d, J=8.59 Hz,
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H), 5.85 (s, 1H), 5.26 (m, 1H), 4.54 (m, 1H), 3.50 (m, 2H), 3.38 (s, 1H), 2.31 (m, 2H), 1.95 (s,
1
3
H), 1.25 (s, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.87 (s, 3H), 0.78 (s, 3H). C NMR (CDCl , 100
3
MHz) δ: 205.4, 193.4, 173.6, 169.7, 144.3, 143.9, 143.8, 142.7, 141.6, 136.4, 124.0, 123.2,
18.6, 111.2, 98.5, 79.8, 74.8, 60.6, 48.1, 48.1, 44.0, 44.0, 43.1, 37.5, 37.4, 37.2, 34.7, 30.4, 29.3,
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1
29.2, 29.1, 29.1, 28.5, 27.6, 26.9, 26.2, 26.1, 25.1, 23.4, 22.8, 21.0 16.4, 15.8, 15.6. HRMS (ESI)
,
+
m/z: [M+H] Calcd for C H N O , 801.4438; found, 801.4405.
4
5
61
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(
3S,7R,8R,10S,13S,17R)-7-acetoxy-17-(furan-3-yl)-4,4,8,10,13-pentamethyl-16-oxo-
2,3,4,5,6,7,8,9,10,11,12,13,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl
nitrobenzo[c][1,2,5]oxadiazol-4-yl)pyrrolidine-2-carboxylate (1c): 40.0 mg (0.056 mmol) of 1c
1-(7-
2
5
was isolated from 30.0 mg (0.066 mmol) of 1 with 85% yield. Orange solid. [α] = +101.2 (c
D
-
1
1
2.25, CHCl ). IR (CHCl ) ν (cm ): 1736, 1701. H NMR (CDCl , 400 MHz) δ: 8.46 (d, J=8.84
3
3
max
3
Hz, 1H), 7.45 (m, 1H), 7.41 (m, 1H), 6.26 (m, 1H), 6.14 (d, J=8.72 Hz, 1H), 5.83 (s, 1H), 5.52
(br. m, 1H), 5.26 (m, 1H), 4.52 (m, 1H), 3.78 (br. m, 2H), 3.37 (s, 1H), 1.93 (s, 3H), 1.23 (s, 3H),
1
3
1
1
.00 (s, 3H), 0.91 (s, 3H), 0.86 (s, 3H), 0.78 (s, 3H). C NMR (CDCl , 100 MHz) δ: 205.4,
3
93.3, 170.5, 169.7, 144.7, 144.3, 143.3, 142.7, 141.6, 135.5, 123.2, 123.1, 118.5, 111.1, 101.7,
82.1, 74.5, 64.7, 60.5, 50.7, 48.1, 48.0, 44.0, 43.0, 37.4, 37.3, 37.2, 31.2, 30.2, 27.6, 26.2, 26.1,
+
2
2.9, 22.7, 21.0, 16.3, 15.7, 15.5. HRMS (ESI) m/z: [M+Na] Calcd for C H N O Na, 737.3162;
3
9
46
4
9
found, 737.3157.
4-allyl-2-methoxyphenyl 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (2a): 77.4
mg (0.176 mmol) of 2a was isolated from 30.0 mg (0.183 mmol) of 2 with 96% yield. Orange
2
5
-1
1
solid. [α] = +0.15 (c 4.1, CHCl ). IR (CHCl ) ν (cm ): 3330, 1754. H NMR (CDCl , 400
D
3
3
max
3
MHz) δ: 8.47 (d, J=8.85 Hz, 1H), 6.92 (d, J=7.93 Hz, 1H), 6.77 (m, 2H), 6.34 (m, 1H), 6.18 (d,
J=8.54 Hz, 1H), 5.96 (m, 1H), 5.11 (m, 2H), 3.79 (s, 3H), 3.54 (dd, J=13.12, 6.17 Hz, 2H), 3.38
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(
m, 2H), 2.64 (t, J=7.32 Hz, 2H), 1.88 (m, 4H), 1.64 (m, 2H). C NMR (CDCl , 100 MHz) δ:
3
1
9
4
71.5, 150.7, 144.2, 143.9, 143.8, 139.1, 137.8, 136.9, 136.4, 124.1, 122.4, 120.7, 116.2, 112.8,
+
8.5, 55.8, 43.7, 40.0, 33.6, 28.1, 26.2, 24.3. HRMS (ESI) m/z: [M+H] Calcd for C H N O ,
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3
4
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7
8
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3
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41.1774; found, 441.1753.
(1R,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl
6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)amino)hexanoate (3a): 79.1 mg (0.184 mmol) of 3a was isolated from 30.0 mg (0.195 mmol)
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-1
of 3 with 95% yield. Orange solid. [α] D = -18.6 (c 3.8, CHCl ). IR (CHCl ) ν (cm ): 3326,
3
3
max
1
1719. H NMR (CDCl , 400 MHz) δ: 8.50 (d, J=8.53 Hz, 1H), 6.40 (m, 1H), 6.18 (d, J=8.78 Hz,
3
1
3
1
H), 4.90 (m, 1H), 3.52 (m, 2H), 2.39 (m, 3H), 0.91 (s, 3H), 0.87 (s, 3H), 0.82 (s, 3H). C NMR
(
CDCl , 100 MHz) δ: 173.7, 144.2, 143.9, 143.8, 136.5, 123.9, 98.5, 79.9, 48.7, 47.8, 44.8, 43.7,
3
+
36.8, 34.2, 28.1, 28.0, 27.1, 26.3, 24.4, 19.6, 18.8, 13.5. HRMS (ESI) m/z: [M+H] Calcd for
C H N O , 431.2294; found, 431.2272.
2
2
31
4
5
(
Z)-5-(2,3-dimethyltricyclo[2.2.1.02,6]heptan-3-yl)-2-methylpent-2-en-1-yl
6-((7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (4a): 62.5 mg (0.126 mmol) of 4a was
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isolated from 30.0 mg (0.136 mmol) of 4 with 93% yield. Orange solid. [α] D = +3.51 (c 1.3,
-
1
1
CHCl ). IR (CHCl ) ν (cm ): 3328, 1719. H NMR (CDCl , 400 MHz) δ: 8.50 (d, J=8.54 Hz,
3
3
max
3
1
2
H), 6.34 (m, 1H), 6.18 (d, J=8.54 Hz, 1H), 5.41 (t, J=7.32 Hz, 1H), 4.61 (s, 2H), 3.52 (m, 2H),
13
.40 (t, J=7.32 Hz, 2H), 1.73 (s, 3H), 0.99 (s, 3H), 0.82 (s, 3H). C NMR (CDCl , 100 MHz) δ:
3
173.4, 144.2, 143.9, 143.8, 136.4, 132.0, 129.0, 124.1, 98.5, 63.2, 45.8, 43.7, 38.1, 34.6, 33.9,
+
3
1.5, 31.0, 28.1, 27.3, 26.3, 24.3, 23.1, 21.4, 19.5, 19.4, 17.5, 10.6. HRMS (ESI) m/z: [M+H]
Calcd for C H N O , 497.2764; found, 497.2740.
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(
1R,2R,5R)-2-methyl-5-(prop-1-en-2-yl)cyclohexyl
6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)amino)hexanoate (5a): 75.2 mg (0.175 mmol) of 5a was isolated from 30.0 mg (0.195 mmol)
2
5
-1
of 5 with 90% yield. Orange solid. [α] = -34.7 (c 2.2, CHCl ). IR (CHCl ) ν (cm ): 3325,
D
3
3
max
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
727. H NMR (CDCl , 400 MHz) δ: 8.49 (d, J=8.72 Hz, 1H), 6.32 (m, 1H), 6.18 (d, J=8.59 Hz,
3
1H), 4.68 (m, 2H), 4.50 (m, 1H), 3.50 (m, 2H), 2.37 (m, 2H), 1.70 (s, 3H), 0.89 (d, J=6.32 Hz,
1
3
3
4
H). C NMR (CDCl , 100 MHz) δ: 173.1, 148.8, 144.2, 143.9, 136.5, 123.8, 108.8, 98.5, 78.3,
3
+
3.7, 43.6, 37.1, 36.9, 34.2, 33.0, 30.8, 28.1, 26.3, 24.4, 20.8, 18.2. HRMS (ESI) m/z: [M+H]
Calcd for C H N O , 431.2294; found, 431.2285.
2
2
31
4
5
(
2S,3R,4S,5R,6R)-2-methoxy-6-(((6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)amino)hexanoyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (6a): 42.2 mg (0.054
2
5
mmol) of 6a was isolated from 30.0 mg (0.059 mmol) of 6 with 91% yield. Orange solid. [α]
=
D
-1
1
-
55.3 (c 2.5, CHCl ). IR (CHCl ) ν (cm ): 3339, 1732. H NMR (CDCl , 400 MHz) δ: 8.43 (d,
3 3 max 3
J=8.70 Hz, 1H), 8.10-7.22 (m, 15H), 6.53 (m, 1H), 6.13 (d, J=8.24 Hz, 1H), 5.97 (t, J=10.08 Hz,
1
4
H), 5.89 (dd, J=10.07, 3.21 Hz, 1H), 5.68 (m, 1H), 5.01 (d, J=1.83 Hz, 1H), 4.42 (dd, J=12.36,
.58 Hz, 1H), 4.33 (m, 2H), 3.54 (s, 3H), 3.47 (m, 2H), 2.37-2.52 (m, 2H), 1.71-1.83 (m, 4H),
1
3
1.50 (m, 2H). C NMR (CDCl , 100 MHz) δ: 173.0, 165.6, 165.4, 165.3, 144.2, 143.9, 143.8,
3
1
9
36.4, 133.5, 133.2, 129.8, 129.7, 129.6, 129.3, 129.0, 128.8, 128.5, 128.5, 128.2, 123.8, 98.6,
+
8.4, 70.4, 69.9, 68.5, 66.8, 62.6, 55.6, 43.6, 33.6, 28.0, 26.2, 24.0. HRMS (ESI) m/z: [M+H]
Calcd for C H N O , 783.2514; found, 783.2508.
4
0
39
4
13
(
7R,8R,10R,13S,17R)-17-(furan-3-yl)-4,4,8,10,13-pentamethyl-3,16-dioxo-
4
,5,6,7,8,9,10,11,12,13,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-7-yl
6-((7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (8a): 35.7 mg (0.052 mmol) of 8a was
25
isolated from 30.0 mg (0.073 mmol) of 8 with 71% yield. Orange solid. [α] D = -66.3 (c 1.6,
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2
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-1
1
CHCl ). IR (CHCl ) ν (cm ): 3307, 1729, 1694, 1667. H NMR (CDCl , 400 MHz) δ: 8.48 (d,
3
3
max
3
J=8.85 Hz, 1H), 7.70 (m, 1H), 7.48 (m, 1H), 7.43 (m, 1H), 7.13 (d, J=10.38 Hz, 1H), 6.25 (m,
1
1
H), 6.19 (d, J=8.85 Hz, 1H), 5.88 (m, 2H), 5.35 (m, 1H), 3.53 (s, 1H), 3.50 (m, 2H), 2.50 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
H), 2.32 (m, 1H), 1.37 (s, 3H), 1.27 (s, 3H), 1.09 (s, 3H), 1.06 (s, 3H), 1.06 (s, 3H). C NMR
(CDCl , 100 MHz) δ: 206.2, 203.9, 193.1, 172.1, 156.6, 144.4, 144.1, 142.9, 141.5, 136.7, 125.8,
3
1
2
23.2, 123.0, 118.1, 111.0, 98.3, 73.8, 60.9, 48.3, 46.1, 44.6, 44.0, 43.7, 40.0, 38.2, 34.1, 29.9,
+
8.1, 26.8, 26.8, 26.4, 26.2, 24.4, 23.4, 21.2, 18.9, 15.7. HRMS (ESI) m/z: [M+H] Calcd for
C H N O , 685.3237; found, 685.3245. Purity (HPLC): 98.5%.
3
8
45
4
8
(
3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-
2
,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl
6-((7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (9a): 49.4 mg (0.075 mmol) of 9a was
25
isolated from 30.0 mg (0.078 mmol) of 9 with 96% yield. Orange solid. [α] D = -20.1 (c 0.9,
-
1
1
CHCl ). IR (CHCl ) ν (cm ): 3331, 1721. H NMR (CDCl , 400 MHz) δ: 8.50 (d, J=8.54 Hz,
3
3
max
3
1
H), 6.28 (m, 1H), 6.18 (d, J=8.54 Hz, 1H), 5.36 (m, 1H), 4.63 (m, 1H), 3.52 (dd, J=13.12, 6.17
Hz, 1H), 2.35 (m, 2H), 2.30 (m, 2H), 1.02 (s, 3H), 0.92 (d, J=6.41 Hz, 3H), 0.88 (d, J=2.41 Hz,
1
3
3H), 0.86 (d, J=2.41 Hz, 3H), 0.68 (s, 3H). C NMR (CDCl , 100 MHz) δ: 172.8, 144.3, 143.9,
3
1
3
43.8, 139.5, 136.4, 124.2, 122.8, 98.5, 74.1, 56.7, 56.1, 50.0, 43.6, 42.3, 39.7, 39.5, 38.2, 36.9,
6.6, 36.2, 35.8, 34.2, 31.9, 31.8, 28.2, 28.2, 28.0, 27.8, 26.3, 24.3, 24.3, 23.8, 22.8, 22.5, 21.0,
+
19.3, 18.7, 11.8. HRMS (ESI) m/z: [M+H] Calcd for C H N O , 663.4485; found, 663.4460.
3
9
59
4
5
1
7-acetyl-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-11-yl 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate
(10a): 48.5 mg (0.080 mmol) of 10a was isolated from 30.0 mg (0.091 mmol) of 10 with 88%
25
-1
yield. Orange solid. [α] = +93.1 (c 1.21, CHCl ). IR (CHCl ) ν (cm ): 3309, 1727, 1701,
D
3
3
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665. H NMR (CDCl , 400 MHz) δ: 8.49 (d, J=8.54 Hz, 1H), 6.66 (m, 1H), 6.19 (d, J=8.54 Hz,
3
H), 5.76 (s, 1H), 5.28 (m, 1H), 3.54 (m, 2H), 2.52 (m, 1H), 2.11 (s, 3H), 1.26 (s, 3H), 0.76 (s,
1
3
H). C NMR (CDCl , 100 MHz) δ: 208.7, 199.2, 172.3, 169.6, 144.2, 143.9, 143.9, 136.5,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
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58
59
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24.7, 123.8, 98.5, 70.7, 62.8, 55.3, 54.7, 45.2, 43.7, 43.6, 39.7, 36.6, 35.0, 34.6, 34.0, 33.2, 31.7,
+
31.2, 28.1, 26.4, 24.2, 24.0, 23.1, 18.3, 14.0. HRMS (ESI) m/z: [M+Na] Calcd for
C H N O Na, 629.2951; found, 629.2945.
3
3
42
4
7
(2'R,4S,5'R,6aR,8aS,8bR,9S,11aS)-5',6a,8a,9-tetramethyl-
1,3,3',4,4',5,5',6,6a,6b,6',7,8,8a,8b,9,11a,12,12a,12b-
icosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-10,2'-pyran]-4-yl
6-((7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (11a): 46.9 mg (0.068 mmol) of 11a was
2
5
isolated from 30.0 mg (0.072 mmol) of 11 with 94% yield. Orange solid. [α] = -64.2 (c 3.2,
D
-1
1
CHCl ). IR (CHCl ) ν (cm ): 3324, 1728. H NMR (CDCl , 400 MHz) δ: 8.49 (d, J=8.72 Hz,
3
3
max
3
1
3
H), 6.29 (m, 1H), 6.18 (d, J=8.59 Hz, 1H), 5.36 (d, J=4.42 Hz, 1H), 4.62 (m, 1H), 4.42 (m, 1H),
.32-3.57 (m, 4H), 2.35 (m, 4H), 1.03 (s, 3H), 0.98 (d, J=6.69 Hz, 3H), 0.80 (s, 3H), 0.79 (d,
13
J=5.49 Hz, 3H). C NMR (CDCl , 100 MHz) δ: 172.8, 144.2, 143.8, 139.5, 136.5, 123.8, 122.4,
3
109.2, 98.5, 80.7, 73.9, 66.8, 62.0, 56.4, 49.9, 43.6, 41.5, 40.2, 39.6, 38.1, 36.9, 36.7, 34.2, 32.0,
3
1.8, 31.3, 30.2, 28.7, 28.1, 27.7, 26.2, 24.3, 20.8, 19.3, 17.1, 16.2, 14.5. HRMS (ESI) m/z:
+
[
M+H] Calcd for C H N O , 691.4071; found, 691.4073 .
39 55
4
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5-methoxy-2-(4-methoxyphenethyl)-1-oxoisoindolin-4-yl 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)amino)hexanoate (12a): 50.8 mg (0.086 mmol) of 12a was isolated from 30.0 mg (0.096
2
5
-
mmol) of 12 with 90% yield. Orange solid. [α] = +0.29 (c 1.48, CHCl ). IR (CHCl ) ν (cm
D
3
3
max
1
1
): 3333, 1759, 1683. H NMR (CDCl , 400 MHz) δ: 8.48 (d, J=8.24 Hz, 1H), 7.24 (d, J=2.29
3
Hz, 1H), 7.13 (d, J=8.70 Hz, 2H), 6.82 (d, J=8.70 Hz, 2H), 6.79 (d, J=1.83 Hz, 1H), 6.50 (m,
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H), 6.18 (d, J=8.70 Hz, 1H), 4.06 (s, 2H), 3.85 (s, 3H), 3.78 (s, 3H), 3.79 (t, J=7.33 Hz, 2H),
.55 (m, 2H), 2.91 (t, J=7.33 Hz, 2H), 2.63 (t, J=7.33 Hz, 2H), 1.80-1.94 (m, 4H), 1.61 (m, 2H).
1
3
C NMR (CDCl , 100 MHz) δ: 170.6, 167.6, 161.0, 158.2, 145.7, 144.2, 143.8, 143.8, 136.4,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
35.7, 130.6, 129.6, 125.0, 124.0, 114.0, 112.4, 104.8, 98.6, 56.0, 55.3, 48.2, 44.5, 43.6, 33.9,
+
33.7, 28.1, 26.3, 24.3. HRMS (ESI) m/z: [M+H] Calcd for C H N O , 590.2251; found,
3
0
32
5
8
5
4
90.2255.
-((2S,3R)-1-(4-fluorophenyl)-3-((S)-3-(4-fluorophenyl)-3-hydroxypropyl)-4-oxoazetidin-2-
yl)phenyl 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (13a): 26.1 mg (0.038
mmol) of 13a was isolated from 30.0 mg (0.073 mmol) of 13 with 52% yield. Orange solid.
2
5
-1
1
[
α] = -25.7 (c 2.5, CHCl ). IR (CHCl ) ν (cm ): 3336, 1743. H NMR (CDCl , 400 MHz) δ:
D 3 3 max 3
8.47 (d, J=8.53 Hz, 1H), 6.90-7.35 (m, 12H), 6.46 (m, 1H), 6.17 (d, J=8.78 Hz, 1H), 4.72 (t,
J=5.77 Hz, 1H), 4.64 (d, J=1.76 Hz, 1H), 3.53 (m, 2H), 3.09 (m, 1H), 2.63 (t, J=7.03 Hz, 2H).
1
3
C NMR (CDCl , 100 MHz) δ: 171.7, 167.3, 162.1 (d, J=245 Hz), 159.0 (d, J=244 Hz), 150.6,
3
1
44.2, 143.8, 139.9, 136.5, 135.1, 133.6, 127.4 (d, J=8.4 Hz), 126.9, 123.9, 122.3, 118.3 (d,
J=7.8 Hz), 115.9 (d, J=22.3 Hz), 115.3 (d, J=20.8 Hz), 98.5, 73.1, 60.7, 60.3, 43.6, 36.5, 33.9,
+
28.1, 26.2, 25.0, 24.2. HRMS (ESI) m/z: [M+Na] Calcd for C H F N O Na, 708.2246; found,
3
6
33
2
5
7
7
08.2222.
(
S)-quinolin-4-yl((1S,2R,4S,5R)-5-vinylquinuclidin-2-yl)methyl
6-((7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (14a): 47.7 mg (0.084 mmol) of 14a was
2
5
isolated from 30.0 mg (0.102 mmol) of 14 with 82% yield. Orange solid. [α] = +43.7 (c 0.9,
D
-
1
1
CHCl ). IR (CHCl ) ν (cm ): 3334, 1734. H NMR (CDCl , 400 MHz) δ: 8.87 (d, J=4.58 Hz,
3
3
max
3
1H), 8.46 (d, J=8.54 Hz, 1H), 8.23 (d, J=8.24 Hz, 1H), 8.11 (d, J=7.93 Hz, 1H), 7.72 (m, 1H),
.61 (m, 1H), 7.37 (d, J=4.58 Hz, 1H), 6.62 (d, J=6.71 Hz, 1H), 6.46 (m, 1H), 6.10 (d, J=8.85
7
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Hz, 1H), 6.01 (m, 1H), 5.11 (m, 2H), 3.36 (m, 3H), 2.95 (m, 2H), 2.82 (m, 1H), 2.74 (m, 1H),
1
3
2
1
9
.46 (m, 2H), 2.30 (q, J=8.24 Hz, 1H). C NMR (CDCl , 100 MHz) δ: 172.3, 149.8, 148.5,
3
45.2, 144.2, 143.9, 143.8, 139.9, 136.4, 130.4, 129.3, 126.9, 125.9, 123.9, 123.3, 118.5, 115.1,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
28
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30
31
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45
46
47
48
49
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51
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59
60
8.5, 73.7, 59.3, 49.7, 49.0, 43.6, 39.5, 33.9, 28.0, 27.7, 26.2, 26.1, 24.1, 23.6. HRMS (ESI) m/z:
+
[M+H] Calcd for C H N O , 571.2669; found, 571.2651.
3
1
35
6
5
(
S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl
-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (15a): 8.1 mg (0.013 mmol) of 15a
6
2
5
was isolated from 50.0 mg (0.144 mmol) of 15 with 09% yield. Orange solid. [α] = -40.4 (c
D
-
1
1
0
1
.6, CHCl ). IR (CHCl ) ν (cm ): 3329, 1744, 1661. H NMR (CDCl , 400 MHz) δ: 8.35 (s,
3 3 max 3
H), 8.31 (d, J=8.85 Hz, 1H), 8.12 (m, 1H), 7.90 (m, 1H), 7.77 (m, 1H), 7.63 (m, 1H), 7.19 (s,
1H), 6.54 (m, 1H), 5.99 (d, J=8.85 Hz, 1H), 5.71 (d, J=17.09 Hz, 1H), 5.42 (d, J=17.09 Hz, 1H),
5
.29 (s, 2H), 3.45 (m, 2H), 2.53-2.65 (m, 2H), 2.25 (m, 1H), 2.14 (m, 1H), 0.99 (t, J=7.63 Hz,
1
3
3
H). C NMR (CDCl , 100 MHz) δ: 172.4, 167.7, 157.3, 152.3, 148.7, 146.2, 145.9, 144.1,
3
1
43.8, 143.8, 136.3, 131.3, 130.7, 129.2, 128.5, 128.3, 128.1, 128.1, 120.1, 98.4, 95.8, 76.0, 67.1,
+
4
9.9, 43.7, 33.4, 31.7, 27.9, 26.0, 24.1, 7.5. HRMS (ESI) m/z: [M+H] Calcd for C H N O ,
3
2
29
6
8
625.2047; found, 625.2044.
(
4R)-2-(2-((1S,2S,6R,8S)-8-((2,2-dimethylbutanoyl)oxy)-2,6-dimethyl-1,2,6,7,8,8a-
hexahydronaphthalen-1-yl)ethyl)-6-oxotetrahydro-2H-pyran-4-yl
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (16a): 40.3 mg (0.058 mmol) of 16a was
6-((7-
2
5
isolated from 30.0 mg (0.072 mmol) of 16 with 81% yield. Orange solid. [α] = +140.0 (c 2.6,
D
-
1
1
CHCl ). IR (CHCl ) ν (cm ): 3323, 1733. H NMR (CDCl , 400 MHz) δ: 8.50 (d, J=8.54 Hz,
3
3
max
3
1H), 6.70 (m, 1H), 6.20 (d, J=8.85 Hz, 1H), 5.99 (d, J=9.77 Hz, 1H), 5.78 (dd, J= 9.77, 6.10 Hz,
H), 5.52 (t, J= 3.36 Hz, 1H), 5.36 (q, J=3.05 Hz, 1H), 5.28 (m, 1H), 4.48 (m, 1H), 3.54 (m, 2H),
1
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5
5
5
6
2
0
1
4
.75 (m, 2H), 2.40 (t, J=7.32 Hz, 2H), 1.13 (s, 3H),, 1.12 (s, 3H), 1.09 (d, J=7.32, 7.02 Hz, 3H),
1
3
.89 (d, J=7.32, 7.02 Hz, 3H), 0.81 (t, J=7.63 Hz, 3H). C NMR (CDCl , 100 MHz) δ: 177.9,
3
72.3, 168.8, 144.3, 144.0, 143.9, 136.5, 132.7, 131.3, 129.8, 128.4, 123.8, 98.5, 77.1, 68.0, 65.7,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
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3
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0
1
2
3
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7
8
9
0
3.7, 43.0, 37.4, 36.7, 35.5, 34.0, 33.3, 33.2, 33.0, 32.8, 30.6, 28.1, 27.2, 26.3, 24.8, 24.7, 24.6,
+
24.4, 23.0, 13.9, 9.3. HRMS (ESI) m/z: [M+Na] Calcd for C H N O Na, 717.3475; found,
3
7
50
4
9
7
17.3434.
(
3S,E)-4-(2-((1R,5R,6R,8aS)-6-hydroxy-5-(hydroxymethyl)-5,8a-dimethyl-2-
methylenedecahydronaphthalen-1-yl)ethylidene)-5-oxotetrahydrofuran-3-yl
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)hexanoate (17a): 17.7 mg (0.028 mmol) of 17a was
6-((7-
2
5
isolated from 30.0 mg (0.086 mmol) of 17 with 33% yield. Orange solid. [α] = -80.7 (c 0.95,
D
-
1
1
CHCl ). IR (CHCl ) ν (cm ): 3409, 1738. H NMR (CDCl , 400 MHz) δ: 8.50 (d, J=8.53 Hz,
3
3
max
3
1
4
H), 7.01 (m, 1H), 6.51 (m, 1H), 6.18 (d, J=8.53 Hz, 1H), 5.94 (d, J=5.52 Hz, 1H), 4.85 (s, 1H),
.56 (m, 1H), 4.47 (s, 1H), 4.23 (m, 1H), 4.15 (d, J=11.04 Hz, 1H), 3.50 (m, 3H), 3.32 (d,
1
3
J=11.04 Hz, 1H), 2.35-2.45 (m, 5H), 1.25 (s, 3H), 0.62 (s, 3H). C NMR (CDCl , 100 MHz) δ:
3
1
72.9, 169.1, 150.6, 146.9, 144.2, 143.9, 143.8, 136.5, 123.9, 123.8, 108.7, 98.6, 80.3, 71.6,
67.8, 64.1, 55.7, 55.0, 43.6, 42.8, 38.7, 37.6, 36.9, 33.7, 29.7, 28.1, 26.3, 25.3, 24.2, 23.6, 22.7,
+
1
2
5.1. HRMS (ESI) m/z: [M+H] Calcd for C H N O , 627.3030; found, 627.3024.
3
2
43
4
9
H,3H-Cyclopenta[b]furo[2',3',4':4,5]naphtho[2,3-d]furan-3,5-diol,
8-(3-furanyl)-
2a,4,5,5a,6,6a,8,9,9a,10a,10b,10c-dodecahydro-6-((6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)amino)hexanoyl)oxy)ethyl-2a,5a,6a,7-tetramethyl-,
(2aR,3R,5S,5aR,6R,6aR,8R,9aR,10aS,10bR,10cR) (18a): 30.2 mg (0.042 mmol) of 18a was
2
5
isolated from 20.0 mg (0.045 mmol) of 18 with 93% yield. Orange solid. [α] = +73.0 (c 0.9,
D
-1
1
CHCl ). IR (CHCl ) ν (cm ): 3140, 1719. H NMR (CDCl , 400 MHz) δ: 8.49 (d, J=8.85 Hz,
3
3
max
3
2
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H), 7.32 (m, 1H), 7.21 (m, 1H), 6.67 (m, 1H), 6.19 (m, 1H), 6.17 (d, J=8.85 Hz, 1H), 5.36 (m,
H), 4.22 (m, 1H), 4.18 (d, J=3.36 Hz, 1H), 3.98 (m, 4H), 3.67 (m, 3H), 3.57 (d, J=7.02 Hz, 1H),
.50 (m, 2H), 3.34 (d, J=6.71 Hz, 1H), 2.61 (d, J=12.82 Hz, 1H), 2.33 (t, J=7.02 Hz, 2H), 1.78
10
11
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53
54
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56
57
58
59
60
13
(
s, 3H), 1.32 (s, 3H), 1.11 (s, 3H), 0.89 (s, 3H). C NMR (CDCl , 100 MHz) δ: 173.3, 148.0,
3
144.2, 144.0, 143.9, 143.1, 138.4, 136.6, 133.4, 127.1, 123.7, 110.4, 98.5, 87.5, 85.9, 77.5, 72.9,
7
2.6, 71.8, 64.8, 49.2, 49.0, 44.3, 43.7, 41.5, 41.5, 38.5, 37.6, 33.8, 31.4, 28.0, 26.2, 24.9, 24.2,
+
1
9.3, 17.1, 15.0, 13.3. HRMS (ESI) m/z: [M+NH ] Calcd for C H N O , 738.3714; found,
4
38 52
5
10
738.3698.
(
3R,5aS,6R,8aS,9R,10R,12S,12aR)-3,6,9-trimethyldecahydro-3H-3,12-
epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl
6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)amino)hexanoate (19a): 42.0 mg (0.075 mmol) of 19a was isolated from 30.0 mg (0.106
25
-1
mmol) of 19 with 71% yield. Orange solid. [α] D = +1.1 (c 0.62, CHCl ). IR (CHCl ) ν (cm ):
3
3
max
1
3
337, 1747. H NMR (CDCl , 400 MHz) δ: 8.50 (d, J=8.53 Hz, 1H), 6.56 (m, 1H), 6.19 (d,
3
J=8.78 Hz, 1H), 5.81 (d, J=9.79 Hz, 1H), 5.45 (s, 1H), 3.53 (m, 2H), 1.41 (s, 3H), 0.98 (d,
13
J=6.02 Hz, 3H), 0.84 (d, J=7.28 Hz, 3H). C NMR (CDCl , 100 MHz) δ: 172.2, 144.2, 143.9,
3
136.5, 123.8, 104.5, 98.5, 91.9, 91.5, 80.1, 51.5, 45.1, 43.6, 37.2, 36.1, 34.0, 33.8, 31.7, 27.9,
+
2
5
6.1, 25.9, 24.5, 23.8, 22.0, 20.2, 12.1. HRMS (ESI) m/z: [M+Na] Calcd for C H N O Na,
2
7
36
4
9
83.2380; found, 583.2370.
(2aR,2a1R,3S,4S,4aR,5S,7aS,8S,10R,10aS)-dimethyl
butyldimethylsilyl)oxy)but-2-yn-1-yl)oxy)-3-hydroxy-4-((1aR,2S,3aS,6aR,7S,7aR)-6a-hydroxy-
a-methyl-1a,2,3a,6a,7,7a-hexahydro-2,7-methanofuro[2,3-b]oxireno[2,3-e]oxepin-1a-yl)-4-
10-acetoxy-5-((4-((tert-
7
methyl-8-(((E)-2-methylbut-2-enoyl)oxy)dodecahydronaphtho[1,8-bc:4,4a-c']difuran-5,10a-
dicarboxylate (20a): 46.7 mg (0.052 mmol) of 20a was isolated from 100.0 mg (0.139 mmol) of
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