Carbocycles related to oseltamivir as influenza virus group-1-specific neuraminidase inhibitors. Binding to N1 enzymes in the context of virus-like particles

Article abstract of DOI:10.1021/jm100822f

We report here the exploitation of the 150-cavity in the active sites of group-1 neuraminidases for the design of new triazole-containing carbocycles related to oseltamivir. Inhibition studies with virus-like particles (VLPs) containing the influenza virus neuraminidase-1 (N1) activity indicate that several candidates are inhibitors, with K_i values in the 10 ^-5-10^-8 M range. In contrast, a known candidate that preserves the free amino group and a new candidate containing a guanidine function are better inhibitors, with K_i values of 1.5 × 10 ^-9 and 4.6 × 10^-10 M, respectively. The most active inhibitor of the N1 enzyme in the triazole series was selective for the N1 class and showed significantly less inhibition (K_i = 2.6 μM vs 0.07 μM) of the free influenza virus neuraminidase-2 (N2). In addition, saturation transfer difference (STD) NMR spectroscopic studies with this compound and the VLPs show that the entire molecule forms contacts with residues in the active site. These data taken together support our proposed binding mode in which the active site and the adjoining 150-cavity are both occupied.

Full text of DOI:10.1021/jm100822f

J. Med. Chem. 2010, 53, 7377–7391 7377
DOI: 10.1021/jm100822f
Carbocycles Related to Oseltamivir as Influenza Virus Group-1-Specific Neuraminidase Inhibitors.
Binding to N1 Enzymes in the Context of Virus-like Particles
Sankar Mohan,† Sarah McAtamney,‡ Thomas Haselhorst,‡ Mark von Itzstein,‡ and Brian Mario Pinto*,†
†Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada, and ‡Institute for Glycomics,
Griffith University (Gold Coast Campus), Southport, Queensland 4222, Australia
Received July 2, 2010
We report here the exploitation of the 150-cavity in the active sites of group-1 neuraminidases for the
design of new triazole-containing carbocycles related to oseltamivir. Inhibition studies with virus-like
particles (VLPs) containing the influenza virus neuraminidase-1 (N1) activity indicate that several can-
didates are inhibitors, with Ki values in the 10-5-10-8 M range. In contrast, a known candidate that
preserves the free amino group and a new candidate containing a guanidine function are better
inhibitors, with Ki values of 1.5ꢀ10-9 and 4.6ꢀ10-10 M, respectively. The most active inhibitor of
the N1 enzyme in the triazole series was selective for the N1 class and showed significantly less inhibition
(Ki = 2.6 μM vs 0.07 μM) of the free influenza virus neuraminidase-2 (N2). In addition, saturation
transfer difference (STD) NMR spectroscopic studies with this compound and the VLPs show that the
entire molecule forms contacts with residues in the active site. These data taken together support our
proposed binding mode in which the active site and the adjoining 150-cavity are both occupied.
Chart 1
Introduction
Influenza A and B viral infections continue to be one of the
serious health problems facing the human population world-
wide. In addition to the seasonal reappearance of the pre-
viously circulated viral strains, the appearance of new viral
strains, such as the swine flu virus (H1N1) now circulating
worldwide, through antigenic variation has resulted in three
major pandemics during the past 100 years. The worst scenario
in the history of influenza outbreaks was the so-called
“Spanish flu” (1918) which killed nearly 1% of the world’s
population. Inhibition of a viral surface glycoprotein called
neuraminidase, which catalyzes the release of newly formed
virions from the infected cells, has proved to be a suitable
approach in the design of antiviral drugs.1-4 This approach
has been successfully demonstrated by the two neuraminidase
inhibitors zanamivir3 and oseltamivir,4 which are currently
in clinical use for the treatment of influenza viral infections
(Chart 1). The worldwide stockpiling of these two antiviral
drugs as part of pandemic preparedness highlights the overall
importance of neuraminidase inhibitors. The alarming threat
of a potential influenza pandemic posed by the avian influenza
virus H5N15,6 and the recent isolations of oseltamivir-resistant
H5N1 underscore the increased demand for the development
of new antiviral drugs.7
The phylogenetic tree divides all of the nine known neur-
aminidase (NAa) subtypes from influenza A into two groups:
group 1 contains N1, N4, N5, and N8 subtypes, whereas
group 2 contains N2, N3, N6, N7, and N9.8 Prior to 2006,
crystal structures of only two subtypes N2 and N9 from
group-2 enzymes were available. In 2006, Russell et al.9
reported the crystal structures of three subtypes, N1, N4,
and N8, from group-1 enzymes. They found that the three-
dimensional shapes of their active sites are virtually identical
but, interestingly, are very different when compared to those
of group-2 enzymes N2 and N9. In the cases of N1, N4, and N8
subtypes, a loop of amino acids consisting of residues
147-152 (also known as 150-loop), including the active site
residues Asp151 and Glu119, was found to adopt an unusual
open conformation compared to the N2 and N9 subtypes in
which the 150-loop was found to have a closed conformation.
As a result of this open-loop conformation, a cavity near the
active site (also called 150-cavity) becomes accessible in the
case of N1, N4, and N8 subtypes. The observation of two
different active-site conformations between certain subtypes
*To whom correspondence should be addressed. Phone: (778) 782-
4152. Fax: (778) 782-4860. E-mail: bpinto@sfu.ca.
aAbbreviations: VLPs, virus-like particles; NA, influenza neuramini-
dase; STD NMR, saturation transfer difference nuclear magnetic reso-
nance; CuAAC, copper-catalyzed azide-alkyne cycloaddition; TLC,
thin layer chromatography; PDC, pyridinium dichromate; DMF, N,N-
dimethylformamide; THF, tetrahydrofuran; DCE, 1,2-dichloroethane;
TFA, trifluoroacetic acid; HEPES, 2-[4-(2-hydroxyethyl)piperazin-
1-yl]ethanesulfonic acid; Neu5Ac2en, 2-deoxy-2,3-dehydro-N-acetylneur-
aminic acid; COSY, correlation spectroscopy; HSQC, heteronuclear
single quantum coherence; MUN, 4-methylumbelliferyl N-acetyl-R-D-
neuraminide.
r
2010 American Chemical Society
Published on Web 09/28/2010
pubs.acs.org/jmc

7378 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Mohan et al.
Figure 1. Active site comparison of group-1 and group-2 enzymes: oseltamivir carboxylate bound in the active site of (A) the N1 subtype and
(B) the N9 subtype.
Figure 2. Active site complex of the N1 subtype with oseltamivir carboxylate showing the C-5 amino group as a potential modification site.
of group-1 and group-2 enzymes suggested that these two
groups are not only genetically distinct but also structurally
distinct.9 Oseltamivir carboxylate (active metabolite of osel-
tamivir, Chart 1) when bound to the N1 subtype showed
similar binding interactions for the ligand as seen in the group-2
enzymes except that the 150-loop adopted the open con-
formation as seen in the apo forms of group-1 enzymes
(Figure 1); however, under certain crystallization conditions,
the 150-loop adopted the closed conformation as seen in apo
and holo forms of group-2 enzymes, suggesting a slow loop
closure upon inhibitor binding.9 Recent computational stud-
ies of the N1 subtype also suggest that the 150-loop has remark-
able mobility and may even open to a greater extent than
observed in the crystal structures.10,11
The flexibility of 150-loop and also the opening of 150-cavity
near the active sites of group-1 enzymes have been considered
as important starting points for the design of group-1 specific
neuraminidase inhibitors.9-11 Although, a universal drug can-
didate against all nine subtypes is highly desirable, group-
1-specific inhibitors that exploit the newly found 150-cavity
would be of use in combating the potential pandemic threat
posed by avian influenza virus H5N1 and alsointhe treatment
of currently circulating swine flu virus H1N1.
One proposed strategy to achieve group-1 specific inhib-
itors is to extend the structures of existing inhibitors by
attaching additional groups of suitable shape, size, and hydro-
phobicity to fill the newly found cavity.9 This action would
presumably also keep the mobile loop in the energetically
favorable open conformation.10,11 Close examination of the
crystal structure of oseltamivir carboxylate bound in the N1
subtype revealed that the C-5 amino group of oseltamivir
carboxylate is well exposed toward the newly discovered
cavity and could serve as the potential modification site in
the design of group-1 specific inhibitors (Figure 2). In this
work, we describe the synthesis of triazole containing carbo-
cycles (1-7, Chart 2) related to oseltamivir as potential group-1
specific neuraminidase inhibitors. The basis of the study was
to investigate the design of selective inhibitors, taking advan-
tage of the novel structural features of influenza virus group-1
neuraminidases. We envisioned that in these compounds
(1-7) the C-40 substituent of the triazole ring could serve as
the cavity filling group and the rigid triazole ring could serve
as the linker between the cavity filling group and oseltamivir-
like scaffold. It has been noted in previous studies that the
C-5 amino group of oseltamivir carboxylate makes key inter-
actions with Asp151 and Glu119 residues in group-2 enzymes
(closed 150-loop conformation).4 However, in the crystal
structures of group-1 enzymes with an open 150-loop con-
formation, the C-5 amino group of oseltamivir is located
further from these residues, and as a result, the C-5 amino
group completely loses its hydrogen bonding interaction with
Asp151.9 Recent computational studies also support this
observation.10 Since the target compounds (1-7) are intended
to exploit the 150-cavity, which indeed requires an open loop
conformation, it was neither necessary nor appropriate to
maintain a basic moiety atthe C-5 positionand hencecould be
substituted with the less basic triazole linker. We anticipated
that while the C-5 amino group interactions observed with the
closed 150-loop would be lost, new interactions in the open
loop region and 150-cavity could be introduced from the tri-
azole moiety and its alkyl substituent that should be compen-
satory. In addition, the triazole linker provides rapid access
to different target compounds with a variety of 150-cavity
binders essentially from one key intermediate. Itisnoteworthy
that similar modification of the zanamivir scaffold has re-
sulted in the discovery of an inhibitor (8, Chart 2) with a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7379
Chart 2
the reported yield of lactone 10 (94%). Hence, we synthesized
compound10 according tothe methodreported for a different
acetal protecting group.14 Thus, D-(-)-quinic acid was treated
with 3,3-dimethoxypentane16 in the presence of a catalytic
amount of p-toluenesulfonic acid (pTsOH) in ethyl acetate.
Continuous removal of ethyl acetate by distillation from the
reaction vessel along with the byproduct methanol led to
complete consumption of quinic acid in 1 h (as indicated by
TLC) and gave the desired lactone 10 in quantitative yield.
The remaining conversion of lactone 10 to the required enone
12 was patterned after the reported procedure. Thus, the
lactone 10 was first converted into the methyl ester 11 using
catalytic NaOMe,14 and pyridinium dichromate (PDC) oxi-
dation followed by elimination gave the desired enone 12
(Scheme 2).15 Subsequent regioselective reduction of the
enone 12 was achieved using NaBH4 at 0 °C to give the allylic
alcohol 13 in 80% yield. The intermediate 13 was then
transformed into the mesylate 14 using MsCl and triethyl-
amine. The mesylate 14, upon reaction with Et3SiH and TiCl4
at -40 °C, underwent regioselective, reductive ring-opening
to give the desired secondary alcohol 15 in 85% yield. An
interesting side product was also formed in this reaction which
was isolated (10% yield) and characterized as the chloride
intermediate 16.17 We predicted the stereochemistry at C-3 in
the chloride 16 as R and the anti relationship between the C-3
chloro substituent and the C-4 hydroxyl group based on the
assumption that the chloride might have formed via SN2
displacement of the mesyl group in 15. The proposed stereo-
chemistry at C-3 was confirmed later by the successful con-
version of the chloride intermediate 16 into an epoxide (17) by
reaction with NaOMe. The mesylate 15 was transformed into
the azido alcohol 18 using sodium azide. Of note, the side
product 16 in the previous step was also transformed into the
desired azido intermediate 18 via a two-step process, namely,
reaction with NaOMe followed by reaction with sodium
azide, as shown in Scheme 2. The azido alcohol 18 was then
reacted with MsCl and triethylamine to give the mesylate19 in
85% yield.
Reduction of the azido group in 19 using PPh3 and triethyl-
amine/water mixture resulted in the formation of the aziridine
compound 20, as shown in Scheme 3. The intermediate 20,
upon reaction with acetyl chloride and triethylamine at 0 °C,
gave the N-acetylaziridine 21. Reaction of 21 with sodium
azide and NH4Cl gave a mixture of three products, as
indicated by TLC (Scheme 3). The major product was isolated
(60% yield) and characterized as the desired azido intermedi-
ate 9. The other two side products could not be separated by
column chromatography. However, we anticipated that these
two side products could have been formed as a result of the
deacetylation of both starting material 21 and product 9.
Hence, the reaction was repeated and when the starting
material was fully consumed (as indicated by TLC), the reac-
tion was stopped, and after processing, the crude product was
treated with acetic anhydride at room temperature for 2 h. TLC
of the reaction mixture indicated only two spots corresponding
to starting material 21 and product 9, thereby confirming that
the two side products observed in the previous step were indeed
the deacetylated products 20 and 22. In this case, the desired
azido intermediate 9 was isolated in 68% yield and the recov-
ered starting material 21 (20%) was then recycled.
Scheme 1
comparable EC50 value (6.4 μM) against avian influenza virus
H5N1 to that of the parent compound, zanamivir (2.8 μM).12
Ensemble-based virtual screening studies indicated that some
of the highest affinity, potential 150-cavity binders interact
primarily through hydrophobic interactions in the 150-cavity.11
Hence, we have chosen variety of lipophilic alkyl groups, from
simple alkyl chains to a complex steroidal group, as 150-cavity
binders in the target compounds (1-7). Included in this list is
also the 1-hydroxypropyl chain of the most potent compound
(8) from the triazole-modified zanamivir series.12
Results and Discussion
Retrosynthetic analysis indicated that the target carbo-
cycles (1-7) could be conveniently synthesized from the azido
intermediate 9 and various terminal alkynes via copper-
catalyzed azide-alkyne cycloaddition (CuAAC)13 as shown
in Scheme 1. For the synthesis of the intermediate 9, we fol-
lowed conceptually similar synthetic strategies that were used
in the synthesis of oseltamivir.4,14 For example, we also
employed reductive ring-opening of a pentylidineketal pro-
tecting group to install the C-5-pentyloxy side chain (step 6
in Scheme 2) and opening of a N-acetylaziridine ring using
sodium azideto install the trans-3,4-diamino functionalityina
regio- and stereospecific manner (step 3 in Scheme 3).
The preparation of the enone intermediate 12 was achieved
using a modified literature procedure starting from D-(-)-
quinic acid as shown in Scheme 2.15 According to the reported
procedure,15 lactone 10 was obtained by heating D-(-)-quinic
acid in boiling 3-pentanone in the presence of a catalytic
amount of phosphoric acid with azeotropic removal of water
for 12 h. However, in our hands, this reaction was found to be
low yielding (25% conversion by TLC) and failed to produce
With the key intermediate 9 in hand, we then performed a
copper-catalyzed azide-alkyne cycloaddition13 using various
terminal alkynes following the standard protocol,18 as shown
in Scheme 4.

7380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Mohan et al.
Scheme 2
Scheme 3
in Scheme 6. In an attempt to provide further evidence for the
proposed mechanism, we have performed the hydrolysis of
compound 25 using deuterated sodium hydroxide in deuter-
ated methanol. As expected, deuterium was incorporated in
the isolated products 3(D), 32(D), and 36(D) at the C-3, C-1,
1
and C-3 positions, respectively, as indicated by H NMR
analyses; the spectrum of compound 32(D) lacked the H-1
signal (Figure 3b) when compared to32(Figure3a). Similarly,
the spectra of compounds 3(D) and 36(D) lacked the H-3
signals, thus indicating that deuterium incorporation had
occurred at C-3 in both of these compounds (data not shown).
In addition, the splitting patterns of the adjacent protons
indicated the incorporation of deuterium. For example, the
1H NMR spectrum of 32 showed a doublet for H-2 at 6.73
ppm and a ddd for both H-6a and H-6b at 2.25 and 1.96 ppm
(Figure 3a), respectively, whereas in the case of 32(D), the
spectrum showed a singlet for H-2 and a dd for both the H-6a
and H-6b protons (Figure 3b). Similarly, the dd correspond-
ing to the H-4 proton observed in the 1H NMR spectrum of 3
(δ 4.20 ppm) and 36 (δ 4.65 ppm) was changed into a doublet
in the spectra of the respective deuterated products 3(D) and
36(D) (data not shown). In a reaction performed in an NMR
tube, the ratio of the three products did not change even after
3 days, indicating that the double bond migration and epi-
merization at the C-3 stereocenter must be occurring prior to
the hydrolysis of the ester group.
To overcome the difficulty of epimerization at C-3 and
double bond migration, we employed trimethyltin hydrox-
ide20 to hydrolyze the methyl esters 27-29. The reactions
proceededsmoothly, andthe productswereobtainedinhigher
yields, as shown in Scheme 7.
Finally, the key intermediate 9 was also converted into the
double bond isomer of oseltamivir 38 and its corresponding
guanidine derivative 39, as shown in Scheme 8. We note that
compound 38 is a known 30 nM inhibitor of neuraminidase.21
Initially, the hydrolysis of the methyl ester 25 was per-
formed using 1 M NaOH and MeOH as solvent (Scheme 5).
1
Surprisingly, the H NMR spectrum of the crude product
indicated three different products. Fortunately, the desired
compound 3 was conveniently precipitated (40% yield, 98%
purity) by the addition of ethyl acetate to the crude product.
From the ethyl acetate soluble fraction, we were able to isolate
one of the side products (41% yield, 96% purity) by crystal-
lization, and it was then characterized as being 32 by 1D and
2D NMR analyses. From the remaining mother liquor, the
other side product was isolated (13% yield) as a mixture
containing 28% of 32 and characterized as being 36. Similar
results were obtained for the hydrolysis of the other esters 23,
24, and 26.19
We attribute the formation of 30-33 and 34-37 to the
base-catalyzed double bond migration and epimerization at
the C-3 stereocenter, respectively, as shown for compound 25

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7381
Scheme 4
Scheme 5
Scheme 6
Thus, intermediate 9, upon treatment with trimethyltin hy-
droxide, gave the intermediate 40 which was then reduced to
theamine 38 by bubbling hydrogengasintothe heterogeneous
mixture of 40 and Lindlar’s catalyst in ethanol. On the other
hand, bubbling hydrogen gas into the heterogeneous mixture
of the intermediate 9 and Lindlar’s catalyst gave compound
41, which was then converted into the Boc-protected guani-
dine derivative 42 using Boc-protected thiourea and HgCl2, as
shown in Scheme 8. The methyl ester 42 was first hydrolyzed
using 1 M KOH, and then the Boc protecting groups were
removed using a 1:1 mixture of trifluoroacetic acid (TFA) and
CH2Cl2 to yield compound 39.
We have tested the inhibitory activities of the target com-
pounds 1-7, 38, and 39 against virus-like particles (VLPs)
that contain an influenza A virus N1 activity.22 Because of the
insoluble nature of compounds 4 and 7 in water, the corre-
sponding L-arginine salts 43 and 44 (Chart 3), respectively,
were made and tested. In order to study the importance of the

7382 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Mohan et al.
Figure 3. 1D traces of 1H NMR spectra of compounds 32 and 32(D).
Scheme 7
N2 subtypes suggests that these triazole-extended carbocycles
indeed take advantage of the 150-cavity found in the N1
subtype for binding.
As a final point of interest, we have used STD NMR
spectroscopy to investigate the ligand interactions with N1
influenza VLPs. Once again, the most active compound in the
triazole series, 2, was chosen as a representative candidate for
1
this study. Figure 4a shows the 1D H NMR spectrum of
a mixture of compound 2 and VLPs containing the N1 sub-
type in deuterated HEPES (2-[4-(2-hydroxyethyl)piperazin-
1-yl]ethanesulfonic acid) buffer. Figure 4b shows the corre-
sponding STD NMR spectrum. Strong STD signals for the
protons corresponding to the oseltamivir-like portion and the
triazole extended portion of the molecule were detected. This
clearly indicates that the entire molecule 2 makes contacts
with the residues in theactive siteof N1-VLPs. Specifically, the
STD signals corresponding to the H-50, H-d, H-e, H-f protons
(STD signals from the triazole-extended portion marked by
the red arrows in Figure 4b) confirm that this portion of the
molecule does indeed make contacts with the receptor molecule
(N1), suggesting that the 150-cavity is also occupied.
Our first series performed as we expected, providing an
excellent leadlike structure that then could be further modified
to increase the potency. Our aim was to demonstrate that we
could access this 150-cavity through the replacement of the
C5-amino group with functionalized triazole substituents on the
oseltamivir-like template and derive selectivity between group-1
and group-2 neuraminidases and maintain high potency. This
has clearly been demonstrated. A lead compound, 2 with a Ki
value of 72 nM and 37-fold group-1 selectivity, has been identi-
fied for further optimization. We also note that the selective
inhibition of group-1 neuraminidases, shown here, has not been
demonstrated previously for the oseltamivir-like template.
In summary, we have synthesized a series of triazole-
containing carbocycles using copper catalyzed azide-alkyne
cycloaddition as a key reaction. One of these compounds, 2,
was shown to be an effective group-1 neuraminidase inhibitor
with a Ki value of 72nM against VLPscontaining an influenza
regiochemistry of the double bond, we have also tested the inhib-
itory activities of compounds 32and 33 as their L-arginine salts
45 and 46 (Chart 3), respectively. The results are summarized
in Table 1.
Our results indicate that all of the triazole modified com-
pounds 1-3, 5, 6, 43, and 44 were inhibitors, with Ki values
ranging from 0.07 to 11 μM (Table 1), but less active than the
parent compound 38, its guanidine derivative 39, or zanami-
vir. The parent compound 38 has a Ki value of 1.5 nM against
the virus-like particles containing an influenza A/N1 activity.
However, its guanidine derivative 39 was found to be a more
potent inhibitor with a Ki value of 0.46 nM. In comparison,
zanamivir has a Ki of 0.16 nM under these assay conditions.
Comparison of the inhibitory activities of 3 and 43 with those
of the corresponding double bond isomers 45 and 46, respec-
tively, leads us to conclude that the presence of a double bond
between C-1 and C-2 is essential for proper orientation of the
triazole ring and its alkyl substituent toward the 150-cavity.
In order to confirm whether the triazole-extended carbo-
cycles (1-3, 5, 6, 43, and 44) make use of the 150-cavity that is
specific to the group-1 subtypes for binding, a representative
compound 2, the most active compound in the triazole series,
was screened against soluble influenza A virus N2, a group-2
neuraminidase in which the 150-cavity is absent. As expected,
compound 2 was found to be significantly less active against the
N2 subtype (Ki = 2.6 μM) compared to the N1 subtype (Ki =
0.07 μM). The observed 37-fold selectivity between N1 and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7383
Scheme 8
Chart 3
is not expected to discriminate between the two groups of
influenza virus neuraminidases on the basis of the structural
comparison of 39 with that of zanamivir.
Experimental Section
General Methods. 1H and 13C NMR spectra were recorded at
600 or 500 and 150 or 125 MHz, respectively. All assignments
were confirmed with the aid of two-dimensional 1H,1H (COSY)
1
and/or H,13C (HSQC) experiments using standard pulse pro-
grams. Processing of the spectra was performed with MestRec
and/or MestReNova software. Analytical thin-layer chroma-
tography (TLC) was performed on aluminum plates precoated
with silica gel 60F-254 as the adsorbent. The developed plates
were air-dried, exposed to UV light and/or sprayed with a solu-
tion containing 1% Ce(SO4)2 and 1.5% molybdic acid in 10%
aqueous H2SO4, and heated. Column chromatography was per-
formed with silica gel 60 (230-400 mesh). High resolution mass
spectra were obtained by the electrospray ionization method,
using an Agilent 6210 TOF LC/MS high resolution magnetic
sector mass spectrometer. The purity of all compounds was
Table 1. Inhibitory Activities of Compounds 1-3, 5, 6, 38, 39, and
43-46 against Virus-like Particles That Contain an Influenza A Virus
N1 Activity
compd
Ki (M)
1
4.6 ꢀ 10-7
7.2 ꢀ 10-8
1.3 ꢀ 10-7
4.8 ꢀ 10-6 a
1.1 ꢀ 10-5 a
1.5 ꢀ 10-9
4.6 ꢀ 10-10
1.2 ꢀ 10-6
5.8 ꢀ 10-6
1.9 ꢀ 10-5
6.0 ꢀ 10-5
5.9 ꢀ 10-6
1.6 ꢀ 10-10
1
determined to be g98% from H and 13C NMR spectra (see
2
Supporting Information).
3
Influenza Virus Sialidase Activity Assay. In a standard 96-well
plate format, by use of previously described virus-like particles
(VLPs) that contain an influenza virus N1 activity22 (kindly
provided by Dr. Jean-Michel Garcia and Jimmy Lai, Hong
Kong University-Pasteur Research Centre, Hong Kong) or
native influenza virus N2 sialidase (kindly provided by Prof.
John Skehel, Mill Hill, U.K.), the synthesized compounds were
assayed for their capacity to inhibit influenza virus sialidase by a
modification23 of the fluorometric method of Potier et al.24
using the fluorogenic substrate 4-methylumbelliferyl N-acetyl-
R-D-neuraminide (MUN). Specifically, 7 μL of 50 mM sodium
acetate-6 mM CaCl2 buffer (pH 5.5) was added to each well of
a 96-well solid black plate on ice, followed by 1 μL of inhibitor,
1 μL of N1-containing VLP or native N2 sialidase, and finally
1 μL of the substrate MUN. The plate was then briefly centri-
fuged up to 1000 rpm for approximately 10 s to combine all
components, and the mixture was incubated at 37 °C with 900 rpm
shaking for 20 min. To stop the reaction, 250 μL of 0.25 M
glycine, pH 10, was added to each well, and the fluorescence was
read (1 s per well) at an excitation of 355 nm and emission of
460 nm. All inhibition assays were done in triplicate over four
inhibitor concentrations and at two concentrations of the sub-
strate MUN (0.05 and 0.1 mM). Inhibitor concentrations were
5
6
38
39
43
44
45
46
Neu5Ac2en
4-deoxy-4-guanidino-Neu5Ac2en
(zanamivir)
a IC50 value.
A virus N1activity. Onthe basis of the observationofselective
inhibition of the N1 subtype by compound 2 and of the ligand
binding studies using STD NMR spectroscopy, it is likely that
the triazole series of compounds (1-3, 5, 6, 43, and 44) make
use of the 150-cavity found in the group-1 subtypes, thus
providing lead structures for further optimization. In addition,
we have also discovered a new candidate 39 (Ki = 0.46 nM)
which has inhibitory activity comparable to that of zanamivir
(Ki = 0.16 nM). Unlike the triazole series, compound 39

7384 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Mohan et al.
Figure 4. (a) 1D 1H NMR spectrum of compound 2 in the presence of H5(N1) influenza VLPs. (b) The corresponding STD NMR spectrum.
selected to give a percentage inhibition of sialidase activity
between 5% and 95%, and data analysis was carried out using
SigmaPlot Enzyme Kinetics Module.25
Methyl (3S,4R,5R)-4,5-O-Isopentylidene-3,4,5-trihydroxycy-
clohexene-1-carboxylate (13). Sodium borohydride (4.5 g, 118.1
mmol) was added slowly (portionwise) to a stirred solution of
compound 12 (6 g, 23.6 mmol) in dry methanol (150 mL) at 0 °C,
and the stirring was continued for 1 h at 0 °C. The reaction was
quenched by the addition of NH4Cl and followed by extraction
with CH2Cl2 (200 mL ꢀ 2). The organic layers were combined
and washed with water (100 mL), brine, dried over anhydrous
Na2SO4, and concentrated. Chromatographic purification (EtOAc/
hexanes, 1:2) of the crude compound afforded compound 13 as
1
STD NMR Analysis. The 1D H NMR spectrum and STD
NMR spectrum of compound 2 (960 μg) in the presence of
H5(N1) influenza VLPs were acquired at 600 MHz and 278 K.
The on-resonance frequency was set to -1.00 ppm and the off-
resonance frequency to 300 ppm. The protein was saturated with
40 Gaussian soft pulses with a duration of 50 ms, each resulting
in a total saturation time of 2.0 s. The residual water signal was
reduced by applying the WATERGATE sequence.
1
a colorless oil (4.9 g, 80%) which solidifies up on storing. H
Methyl 4,5-O-Isopentylidene-3-dehydro-4-epi-shikimate (12).
To a stirred solution of D-(-)-quinic acid (10 g, 52.1 mmol) in
ethyl acetate (250 mL) was added 3,3-dimethoxypentane16 (20.7g,
156.3 mmol) and a catalytic amount of p-toluenesulfonic acid
(100 mg). The reaction mixture was subjected to slow distillation.
Continuous removal of ethyl acetate (∼125 mL) from the reac-
tion vessel along with the byproduct methanol led to complete
consumption of starting material in 1 h (as indicated by TLC).
The reaction mixture was then concentrated under reduced
pressure to give the lactone 10 in quantitative yield. To the crude
lactone 10 was added a solution of sodium methoxide (280 mg,
5.2 mmol) in 200 mL of methanol. After stirring at room tem-
perature for 2 h, the reaction mixture was stored at -20 °C for
16 h. The mixture was then neutralized with glacial acetic acid
(0.5 mL) at -20 °C. All of the volatile components were removed
under reduced pressure and the resulting crude mixture was
purified by column chromatography (EtOAc/hexanes 4:1) to give
11 as a pale yellow oil (13.5 g, 94%). This material was identical in
all respects to that reported previously.15 The methyl ester 11 was
then transformed into the enone 12 using a reported procedure.15
NMR (CDCl3, 600 MHz): δ 6.91 (1H, br dddd-like, H-2), 4.61
(1H, ddd, J5,6a = 2.4, J5,6b = 3.6, J5,4 = 7.8 Hz, H-5), 4.54 (1H,
ddd, J4,3 = 4.8, J4,2 = 1.5 Hz, H-4), 4.06 (1H, dddd, J3,6b =
J
3,2 = 2.4, J3,-OH = 10.2 Hz, H-3), 3.74 (3H, s, -COOCH3), 3.07
(1H, ddd, J6a,6b = 16.8, J6a,2 = 0.6 Hz, H-6a), 2.72 (1H, d, -OH),
1.91 (1H, dddd, J6b,2=2.4 Hz, H-6b), 1.61-1.53 (4H, m, (CH3-
CH2)2-), 0.86 and 0.73 (3H, t, J = 7.8 Hz, (CH3CH2)2-). 13C
NMR (CDCl3, 150 MHz): δ 166.3 (-COOCH3), 143.1 (C-2),
128.6 (C-1), 113.0 ((R)2-C-(OR)2), 76.4 (C-4), 72.6 (C-5), 68.5
(C-3), 52.2 (COOCH3), 29.1 and 28.3 (CH3CH2)2-), 26.7 (C-6),
8.9 and 7.8 (CH3CH2)2-). HRMS Calcd for C13H21O5 (M þ H):
257.1389. Found: 257.1393.
Methyl (3S,4R,5R)-4,5-O-Isopentylidene-3-O-(methanesulfonyl)-
3,4,5-trihydroxycyclohex-1-ene-1-carboxylate (14). A solution
of methanesulfonyl chloride (3.82 mL, 49.2 mmol) in 10 mL of
CH2Cl2 was added dropwise to a stirred solution of compound
13 (6.0 g, 23.4 mmol) and Et3N (8.48 mL, 60.9 mmol) in CH2Cl2
(160 mL) at 0 °C. After the addition, the reaction mix-
ture was stirred at room temperature for 5 h. The reaction
mixture was then poured into ice cold water and extracted with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7385
CH2Cl2 (100 mL ꢀ 2). Combined organic layer was washed with
brine, dried over anhydrous Na2SO4, and concentrated. Chro-
matographic purification (EtOAc/hexanes, 1:4) of the crude
compound afforded compound 14 as a pale yellow oil (7.4 g,
94%). However, pure mesylate 14 was found to be unstable (as
indicated by 1H and 13C NMR), and hence, it was taken to the
next step immediately. 1H NMR (CDCl3, 500 MHz): δ 6.92 (1H,
-COOCH3), 3.58 (1H, ddd, J4,3 = 4.2, J4,6a=1.8 Hz, H-4), 3.42
(1H, dd, H-3), 3.31 (1H, m, (CH3CH2)2CH-O-), 2.80 (1H, ddd,
J
6a,6b=16.8 Hz, H-6a), 2.14 (1H, ddd, H-6b), 1.55-1.46 (4H, m,
(CH3CH2)2-), 0.92 and 0.90 (3H, t, J=7.8 Hz, (CH3CH2)2-). 13
C
NMR (CDCl3, 150 MHz): δ 166.4 (-COOCH3), 134.0 (C-1),
133.8 (C-2), 81.6 ((CH3CH2)2CH-O-), 73.1 (C-5), 57.5 (C-4),
52.3 (COOCH3), 48.6 (C-3), 27.3 (C-6), 26.85 and 26.77 ((CH3-
CH2)2CH-O-), 10.0 and 9.8 ((CH3CH2)2CH-O-). HRMS Calcd
for C13H21O4 (MþH): 241.1440. Found: 241.1444.
br dddd-like, H-2), 5.03 (1H, ddd, J3,6b = 2.4, J3,4 = 4.0, J3,2
=
2.0 Hz, H-3), 4.73 (1H, ddd, J4,5=7.5, J4,2=2.0 Hz, H-4), 4.63
(1H, ddd, J5,6a = 2.4, J5,6b = 4.0 Hz, H-5), 3.76 (3H, s,
-COOCH3), 3.20 (3H, s, -OMs), 3.10 (1H, br ddd-like, J6a,6b=
17.0 Hz, H-6a), 1.93 (1H, br dddd-like, J6b,2 = 3.0 Hz, H-6b),
1.65-1.53 (4H, m, (CH3CH2)2-), 0.86 and 0.73 (3H, t, J=7.8 Hz,
(CH3CH2)2-). 13C NMR (CDCl3, 150 MHz): δ 165.5 (-COO-
CH3), 136.7 (C-2), 130.8 (C-1), 113.7 ((R)2-C-(OR)2), 76.3 (C-3),
75.5 (C-4), 72.7 (C-5), 52.4 (-COOCH3), 39.3 (-OMs), 29.0 and
28.1 (CH3CH2)2-), 27.4 (C-6), 8.9 and 7.6 (CH3CH2)2-).
Methyl (3R,4S,5R)-3-Azido-5-(1-ethylpropoxy)-4-hydroxycyclo-
hex-1-ene-1-carboxylate (18). To a stirred solution of compound
15 (22.5 g, 66.9 mmol) in dry DMF (600 mL) was added sodium
azide (19.5 g, 301 mmol), and the resulting mixture was stirred at
room temperature for 16 h. Note: The following workup was done
in four small batches, and combined crude material was taken for
purification. Heating of the material at above 30 °C was avoided at
anytime during the workup and purification process as a safety
precaution. The reaction mixture was diluted with ethyl acetate
(200 mL) and washed with saturated NH4Cl, water, dried over
Na2SO4, and solvents were removed at 30 °C under reduced
pressure. The combined crude material was purified by column
chromatography (EtOAc/hexanes 1:3) to give compound 18
(16.9 g, 89%) as a colorless oil. 1H NMR (CDCl3, 600 MHz): δ
6.70 (1H, ddd, J2,3=3.0, J2,6a=1.2, J2,6b=2.4 Hz, H-2), 4.21 (1H,
dddd, J3,4=7.2, J3,6a=1.8, J3,6b=2.4 Hz, H-3), 3.84 (1H, ddd,
Methyl (3S,4R,5R)-5-(1-Ethylpropoxy)-4-hydroxy-3-(metha-
nesulfonyloxy)cyclohex-1-ene-1-carboxylate (15). To a stirred
solution of compound 14 (27 g, 80.8 mmol) in CH2Cl2 (600 mL)
at -40 °C, triethylsilane (15.5 mL, 97.0 mmol) was added slowly
followed by the dropwise addition of TiCl4 (9.8 mL, 89.7 mmol)
dissolved in 150 mL of CH2Cl2. The reaction temperature was
maintained at -40 °C throughout the addition of TiCl4 which
was completed in 1.5 h. The reaction mixture was then poured
into ice cold water (500 mL) and stirred for 10 min. The aqueous
layer from the resulting biphasic layer was removed. The organic
layer was washed with aqueous NaHCO3 (200 mL ꢀ 2), dried
over anhydrous Na2SO4, and all of the volatile components were
removed under reduced pressure. The crude compound was
purified by column chromatography (EtOAc/hexanes 1:3) to
give the desired compound 15 as a pale yellow oil (23.3 g,
85%) and also the side product, chloride 16, as a colorless oil
(2.7 g, 10%). However, chloride 16 was found to be unstable,
and hence, only 1H NMR and HRMS data were obtained. Data
for 15: 1H NMR (CDCl3, 600 MHz) δ 6.67 (1H, br dddd-like,
H-2), 5.33 (1H, br dddd-like, H-3), 4.30 (1H, ddd, J4,3 = 4.2,
J4,5 = J4,2 =1.8 Hz, H-4), 3.74 (3H, s, -COOCH3), 3.62 (1H,
ddd, J5,6a=6.0, J5,6b=9.0 Hz, H-5), 3.26 (1H, m, (CH3CH2)2-
CH-O-), 3.13 (3H, s, -OMs), 2.62 (1H, br dddd-like, J6a,6b=18.0,
J6a,3=J6a,2=1.2 Hz, H-6a), 2.47 (1H, dddd, J6b,3=J6b,2=3.0 Hz,
H-6b), 1.54-1.42 (4H, m, (CH3CH2)2CH-O-), 0.89 and 0.87
(3H, t, J = 7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CDCl3, 150
MHz): δ 166.1 (-COOCH3), 132.4 (C-2), 132.3 (C-1), 81.0 (CH3-
CH2)2CH-O-), 76.4 (C-3), 73.1 (C-5), 68.3 (C-4), 52.4 (-COO-
CH3), 39.3 (-OMs), 27.0 (C-6), 26.5 and 26.4 (CH3CH2)2CH-O-),
9.9 and 9.7 (CH3CH2)2CH-O-). Anal. Calcd for C14H24O7S: C,
49.98; H, 7.19. Found: C, 49.96; H, 7.30.
J5,6a=J5,6b=4.2, J5,4=3.0 Hz, H-5), 3.74 (3H, s, -COOCH3), 3.72
(1H, ddd, J4,-OH=7.8 Hz, H-4), 3.27 (1H, m, (CH3CH2)2CH-O-),
2.62 (1H, dddd, J6a,6b=18.6 Hz, H-6a), 2.43 (1H, dddd, H-6b),
2.36 (1H, d, -OH), 1.58-1.34 (4H, m, (CH3CH2)2CH-O-), 0.88
and 0.80 (3H, t, J = 7.8 Hz, (CH3CH2)2CH-O-). 13C NMR
(CDCl3, 150 MHz): δ 166.6 (-COOCH3), 133.8 (C-2), 130.3 (C-1),
80.3 (CH3CH2)2CH-O-), 73.0 (C-5), 72.4 (C-4), 61.6 (C-3), 52.3
(-COOCH3), 28.6 (C-6), 26.7 and 26.3 (CH3CH2)2CH-O-), 9.9
and 9.7 (CH3CH2)2CH-O-). HRMS Calcd for C13H22N3O4 (M þ
H): 284.1610. Found: 284.1602.
Methyl (3R,4S,5R)-3-Azido-5-(1-ethylpropoxy)-4-(methane-
sulfonyloxy)cyclohex-1-ene-1-carboxylate (19). A solution of
methanesulfonyl chloride (11.2 mL, 144.1 mmol) in 50 mL of
CH2Cl2 was added dropwise to a stirred solution of compound
18 (19.4 g, 68.6 mmol) and Et3N (24.8 mL, 178.4 mmol) in
CH2Cl2 (550 mL) at 0 °C. After the addition, the reaction
mixture was stirred at 0 °C for 1 h. Note: The following workup
was done in four small batches, and combined crude material was
taken for purification. Heating of the material at above 30 °C was
avoided at anytime during the workup and purification process as a
safety precaution. The reaction mixture was poured into ice cold
water and extracted with CH2Cl2 (100 mL ꢀ 2). Combined
organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated at 25 °C under reduced pressure.
Chromatographic purification (EtOAc/hexanes, 1:3) of the
combined crude material afforded compound 19 (21.1 g, 85%)
as a pale yellow oil. 1H NMR (CDCl3, 600 MHz): δ 6.73 (1H,
Methyl (3R,4R,5R)-3-Chloro-5-(1-ethylpropoxy)-4-hydroxy-
cyclohex-1-ene-1-carboxylate (16). 1H NMR (CDCl3, 500 MHz):
δ 6.81 (1H, ddd, J2,3 = 4.0, J2,6a = J2,6b = 2.0 Hz, H-2), 4.62
(1H, dddd, J3,4 = 5.5, J3,6a = J3,6b = 2.0 Hz, H-3), 4.0 (1H, dd,
ddd, J2,3=2.4, J2,6a=1.2, J2,6b=3.0 Hz, H-2), 4.58 (1H, dd, J4,3
=
J4,5 = 2.5 Hz, H-4), 3.91 (1H, ddd, J5,6a = 5.5, J5,6b = 6.5 Hz,
7.2, J4,5=2.4 Hz, H-4), 4.52 (1H, dddd, J3,6a=1.8, J3,6b=3.0 Hz,
H-3), 4.01 (1H, ddd, J5,6a =J5,6b =4.2 Hz, H-5), 3.75 (3H, s,
-COOCH3), 3.32 (1H, m, (CH3CH2)2CH-O-), 3.11 (3H, s,
-OMs), 2.62 (1H, dddd, J6a,6b=18.6 Hz, H-6a), 2.52 (1H, dddd,
H-6b), 1.54-1.36 (4H, m, (CH3CH2)2CH-O-), 0.88 and 0.80
(3H, t, J=7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CDCl3, 150
MHz): δ 166.1 (-COOCH3), 132.4 (C-2), 131.1 (C-1), 81.8 (CH3-
CH2)2CH-O-), 81.1 (C-4), 71.9 (C-5), 59.0 (C-3), 52.4 (-COO-
CH3), 38.6 (-OMs), 29.8 (C-6), 26.4 and 26.1 (CH3CH2)2CH-O-),
9.8 and 9.5 (CH3CH2)2CH-O-). HRMS Calcd for C14H24N3O6S
(M þ H): 362.1386. Found: 362.1363. C14H23N3NaO6S (M þ
Na): 384.1205. Found: 384.1200. C14H23KN3O6S (M þ K):
400.0945. Found: 400.0944.
H-5), 3.75 (3H, s, -COOCH3), 3.30 (1H, m, (CH3CH2)2CH-O-),
2.58 (1H, dddd, J6a,6b = 18.5 Hz, H-6a), 2.52 (1H, dddd, H-6b),
2.40 (1H, br s, -OH), 1.57-1.38 (4H, m, (CH3CH2)2CH-O-),
0.89 and 0.84 (3H, t, J = 7.8 Hz, (CH3CH2)2CH-O-). HRMS
Calcd for C13H22ClO4 (M þ H): 277.1207. Found: 277.1233.
Methyl (3S,4R,5R)-3,4-Epoxy-5-(1-ethylpropoxy)cyclohex-1-
ene-1-carboxylate (17). To a stirred solution of chloride 16 (560
mg, 2.03 mmol) in dry methanol (25 mL) at room temperature,
sodium methoxide (219.2 mg, 4.06 mmol) was added, and the
stirring was continued for 18 h. The mixture was then diluted
with CH2Cl2 (75 mL) and washed with water (50 mL ꢀ 2). The
organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated to dryness. The crude material was purified by
column chromatography (EtOAc/hexanes 1:4) to give the ep-
oxide 17 (299 mg, 61%) as a colorless oil. 1H NMR (CDCl3, 600
MHz): δ 6.96 (1H, dd, J2,3 = J2,6b = 3.6 Hz, H-2), 3.80 (1H,
ddd, J5,4 =1.2, J5,6a =6.6, J5,6b =10.8 Hz, H-5), 3.71 (3H, s,
Methyl (3R,4R,5R)-3,4-(Acetylepimino)-5-(1-ethyl-propoxy)-
cyclohex-1-ene-1-carboxylate (21). A solution of compound 19
(22.1 g, 61.2 mmol) and triphenylphosphine (17.6 g, 67.3 mmol)
in THF (1 L) was stirred at room temperature. After 2 h of

7386 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Mohan et al.
stirring, water (100 mL) was added followed by the addition of
Et3N (25.3 mL, 183.6 mmol). Stirring was continued for 14 h.
Solvent was evaporated, and the residue was dissolved in CH2-
Cl2 (300 mL) and washed with water (150 mL ꢀ 2). The organic
layer was dried over Na2SO4, filtered, and concentrated. At-
tempts to remove the triphenylphosphine oxide byproduct by
column chromatography were unsuccessful. Hence, the crude
compound was suspended in ether (400 mL) and stirred for 2 h.
The separated triphenylphosphine oxide was removed by filtra-
tion. The filtrate was then concentrated and resuspended in
50 mL of ether and cooled to -10 °C for 2 h. Once again, the
separated solid was removed by filtration and the filtrate was
concentrated to give a 9:1 (14.3 g) mixture of aziridine com-
pound 20 and triphenylphosphine oxide. This mixture was dis-
solved in CH2Cl2 (650 mL) and stirred at 0 °C. Et3N (48.8 mL,
351.0 mmol) was then added followed by the dropwise addition
of freshly distilled acetyl chloride (12.5 mL, 175.7 mmol). The
reaction temperature was raised to room temperature, and stirr-
ing was continued for 0.5 h. The reaction mixture was diluted
with ethyl acetate and washed with water (200 mL ꢀ 2). The
organic layer was dried over anhydrous Na2SO4, concentrated,
and column-purified (EtOAc/hexanes 1:4) to give compound 21
(10.2 g, 59% for two steps) as a pale yellow oil. 1H NMR
went to completion after vigorous stirring at room temperature for
2 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and
washed with 10% NH4OH (50 mL ꢀ 2) and water (50 mL ꢀ 2).
The organic layer was dried over anhydrous Na2SO4, concen-
trated to dryness, and column-purified (MeOH/CHCl3 1:9) to
give compound 23 (116 mg, 92%) as a yellow foam. 1H NMR
(CDCl3, 500 MHz): δ 7.40 (1H, s, H-50), 6.73 (1H, br dd, J2,3
2.5, J2,6b=3.0 Hz, H-2), 5.93 (1H, br dddd, J3,4 = 10.0, J3,6a
=
=
2.0, J3,6b = 4.0 Hz, H-3), 5.92 (1H, d, J-NH,4 = 8.0 Hz, -NHAc),
4.08 (1H, ddd, J5,4 = 10.0, J5,6a = 5.5, J5,6b = 9.5 Hz, H-5), 3.94
(1H, ddd, H-4), 3.75 (3H, s, -COOCH3), 3.64 (2H, m,
HOCH2CH2CH2-), 3.31 (1H, m, (CH3CH2)2CH-O-), 3.01
(1H, br ddd, J6a,6b = 18.0 Hz, H-6a), 2.80 (2H, dd, J = 7.8
Hz, HOCH2CH2CH2-), 2.36 (1H, dddd, H-6b), 2.0 (br s, -OH),
1.90 (2H, m, HOCH2CH2CH2-), 1.86 (3H, s, -NHCOCH3),
1.54-1.40 (4H, m, (CH3CH2)2CH-O-), 0.87 and 0.83 (3H, t,
J = 7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CDCl3, 125 MHz):
δ 171.3 (-NHCOCH3), 166.0 (-COOCH3), 147.7 (C-40), 134.0
(C-2), 131.2 (C-1), 121.4 (C-50), 81.6 (CH3CH2)2CH-O-), 72.6
(C-5), 61.0 (HOCH2CH2CH2-), 60.8 (C-3), 56.0 (C-4), 52.3
(-COOCH3), 32.1 (C-6), 31.9 (HOCH2CH2CH2-), 26.2 and
25.7 (CH3CH2)2CH-O-), 23.1 (-NHCOCH3), 21.9 (HOCH2-
CH2CH2-), 9.5 and 9.2 (CH3-CH2)2-CH-O-). HRMS Calcd
for C20H33N4O5 (MþH): 409.2451. Found: 409.2452. C20H32-
N4NaO5 (M þ Na): 431.2270. Found: 431.2266.
(CDCl3, 600 MHz): δ 7.17 (1H, ddd, J2,3=3.0, J2,6a=1.8, J2,6b
=
3.6 Hz, H-2), 4.20 (1H, ddd, J5,4 =J5,6a =2.4, J5,6b =3.0 Hz,
H-5), 3.73 (3H, s, -COOCH3), 3.23 (1H, m, (CH3CH2)2CH-O-),
3.12-3.09 (2H, m, H-3, H-4), 2.90 (1H, ddd, J6a,6b =16.8 Hz,
H-6a), 2.12 (3H, s, -NHCOCH3), 2.06 (1H, ddd, H-6b), 1.52-
1.32 (4H, m, (CH3CH2)2CH-O-), 0.88 and 0.78 (3H, t, J=7.8
Hz, (CH3CH2)2CH-O-). 13C NMR (CDCl3, 150 MHz): δ 181.8
(-NHCOCH3), 166.8 (-COOCH3), 133.4 (C-2), 130.5 (C-1), 81.3
(CH3CH2)2CH-O-), 68.1 (C-5), 52.2 (-COOCH3), 41.2 (C-3)*,
33.1 (C-4)*, 26.9 and 26.7 (CH3CH2)2CH-O-), 26.4 (C-6), 23.5
(-NHCOCH3), 10.1 and 9.7 (CH3CH2)2CH-O-), where / indi-
cates interchangeable. HRMS Calcd for C15H24NO4 (M þ H):
282.1705. Found: 282.1704.
Methyl (3S,4R,5R)-4-Acetamido-3-azido-5-(1-ethylpropoxy)-
cyclohex-1-ene-1-carboxylate (9). To a stirred solution of com-
pound 21 (10.0 g, 35.6 mmol) in dry DMF (350 mL) was added
sodium azide (11.6 g, 177.9 mmol) and NH4Cl (7.5 g, 142.4
mmol). The resulting mixture was stirred at room temperature
for 16 h. The reaction mixture was diluted with ethyl acetate
(200 mL) and washed with water, dried over Na2SO4, and all of
the volatile components were removed at 30 °C under reduced
pressure. The crude compound was dissolved in acetic anhy-
dride (75 mL) and stirred for 2 h. Acetic anhydride was removed
carefully under reduced pressure. The resulting crude material
was purified by column chromatography (EtOAc/hexanes 1:2)
to give compound 9 (7.8 g, 68%) as a yellow oil which solidifies
up on storing. 1H NMR (CDCl3, 500 MHz): δ 6.69 (1H, br dd,
Methyl (3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(1-
hydroxypropyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxylate (24).
Compound 24 (pale yellow foam, 114 mg, 90%) was obtained as
a 1:1 mixture of diastereomers from compound 9 (100 mg, 0.31
mmol) and pent-1-yn-3-ol (34.7 μM, 0.40 mmol) using the
procedure as that described to obtain 23. Data for the mixture
1
of diastereomers: H NMR (CDCl3, 500 MHz) δ 7.53 (2H, s,
H-50), 6.74 and 6.73 (1H, br dd, J2,3 = 2.5, J2,6b = 3.0 Hz, H-2),
6.0 (2H, br d, J3,4 = 10.0 Hz, H-3), 5.96 (2H, d, J-NH,4 = 7.5 Hz,
-NHAc), 4.77 (2H, dd, CH3CH2CH(OH)-), 4.15 and 4.14 (1H,
ddd, J5,4 = 10.0, J5,6a = 5.5, J5,6b = 10.0 Hz, H-5), 3.87 and 3.86
(1H, ddd, H-4), 3.75 (6H, s, -COOCH3), 3.31 (2H, m, (CH3-
CH2)2CH-O-), 3.0 (2H, br dddd-like, J6a,6b = 18.0 Hz, H-6a),
2.36 (2H, dddd, J6b,2 = J6b,3 = 3.0 Hz, H-6b), 2.10 (br s, -OH),
1.92-1.80 (4H, m, CH3CH2CH(OH)-), 1.86 and 1.85 (3H, s,
-NHCOCH3), 1.54-1.40 (8H, m, (CH3CH2)2CH-O-), 0.95 (6H, t,
J = 7.5 Hz, CH3CH2CH(OH)-), 0.88 and 0.83 (6H, t, J = 7.5
Hz, (CH3CH2)2CH-O-). 13C NMR (CDCl3, 125 MHz): δ 171.5
and 171.4 (-NHCOCH3), 166.13 and 166.12 (-COOCH3), 151.9
and 151.8 (C-40), 134.1 and 134.0 (C-2), 131.2 and 131.1 (C-1),
122.0 and 121.8 (C-50), 81.6 (CH3CH2)2CH-O-), 72.5 (C-5), 68.2
and 68.1 (CH3CH2CH(OH)-), 60.9 and 60.8 (C-3), 56.4 (C-4),
52.38 and 52.36 (-COOCH3), 32.2 (C-6), 30.5 and 30.4 (CH3-
CH2CH(OH)-), 26.3 and 25.8 (CH3CH2)2CH-O-), 23.3 and 23.2
(-NHCOCH3), 10.0 and 9.9 (CH3CH2CH(OH)-), 9.57, 9.56,
9.30, and 9.29 (CH3-CH2)2-CH-O-). HRMS Calcd for C20H33-
N4O5 (M þ H): 409.2451. Found: 409.2452. C20H32N4NaO5
(M þ Na): 431.2270. Found: 431.2266.
J2,3=2.5, J2,6b=3.0, H-2), 5.64 (1H, d, J-NH,4=7.5 Hz, -NHAc),
4.77 (1H, dddd-like, J3,4=8.5 Hz, H-3), 4.03 (1H, ddd, J5,4
=
10.0, J5,6a=5.5, J5,6b=9.0 Hz, H-5), 3.75 (3H, s, -COOCH3),
3.39 (1H, ddd, H-4), 3.27 (1H, m, (CH3CH2)2CH-O-), 2.86 (1H,
br dddd-like, J6a,6b=18.0 Hz, H-6a), 2.19 (1H, dddd, J6b,3=3.5,
Methyl (3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(1-
hydroxy-1-methylethyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-car-
boxylate (25). Compound 25 (385 mg, 88%) was obtained as
a colorless foam from compound 9 (350 mg, 1.08 mmol) and
2-methylbut-3-yn-2-ol (0.14 mL, 1.40 mmol) using the proce-
dure as that described to obtain 23. 1H NMR (CDCl3, 500
J6b,2 = 3.0 Hz, H-6b), 2.00 (3H, s, -NHCOCH3), 1.53-1.38
(4H, m, (CH3CH2)2CH-O-), 0.86 and 0.85 (3H, t, J=7.5 Hz,
(CH3CH2)2CH-O-). 13C NMR (CDCl3, 150 MHz): δ 171.2
(-NHCOCH3), 166.4 (-COOH), 134.6 (C-2), 130.7 (C-1), 81.2
(CH3CH2)2CH-O-), 71.0 (C-5), 59.7 (C-3), 57.8 (C-4), 52.4
(-COOCH3), 31.5 (C-6), 26.4 and 25.9 (CH3CH2)2CH-O-),
24.0 (-NHCOCH3), 9.8 and 9.4 (CH3CH2)2CH-O-). HRMS
Calcd for C15H25N4O4 (M þ H): 325.1876. Found: 325.1878.
Methyl (3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(3-
hydroxypropyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxylate (23).
To a stirred solution of compound 9 (100 mg, 0.31 mmol) and
pent-4-yn-1-ol (37.5 μL, 0.40 mmol) in a 1:1 mixture (2 mL) of
water and tert-butanol, copper(II) sulfate pentahydrate (5.4 mg)
was added followed by the addition of freshly prepared 1 M
solution (0.11 mL) of sodium ascorbate in water. The reaction
MHz): δ 7.49 (1H, s, H-50), 6.75 (1H, br dd, J2,3 = J2,6b
2.5 Hz, H-2), 6.01 (1H, dddd, J3,4 = 9.5, J3,6a = 1.5, J3,6b
=
=
3.0 Hz, H-3), 5.69 (1H, d, J-NH,4=7.5 Hz, -NHAc), 4.16 (1H,
ddd, J5,4=9.5, J5,6a=5.5, J5,6b=10.0 Hz, H-5), 3.86 (1H, ddd,
H-4), 3.75 (3H, s, -COOCH3), 3.31 (1H, m, (CH3CH2)2CH-O-),
2.99 (1H, br ddd, J6a,6b=18.0 Hz, H-6a), 2.37 (1H, dddd, H-6b),
1.87 (3H, s, -NHCOCH3), 1.60 (6H, s, (CH3)2C(OH)-), 1.53-
1.40 (4H, m, (CH3CH2)2CH-O-), 0.88 and 0.82 (3H, t, J=7.8
Hz, (CH3CH2)2CH-O-). 13C NMR (CDCl3, 125 MHz): δ 171.3
(-NHCOCH3), 166.1 (-COOCH3), 156.2 (C-40), 134.1 (C-2),
131.1 (C-1), 121.1 (C-50), 81.5 (CH3CH2)2CH-O-), 72.7 (C-5),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7387
68.4 ((CH3)2C(OH)-), 60.9 (C-3), 56.2 (C-4), 52.3 (-COOCH3),
32.2 (C-6), 30.7 and 30.4 ((CH3)2C(OH)-), 26.2 and 25.8
(CH3CH2)2CH-O-), 23.2 (-NHCOCH3), 9.6 and 9.2 (CH3CH2)2-
CH-O-). HRMS Calcd for C20H33N4O5 (M þ H): 409.2451.
Found: 409.2453. C20H32N4NaO5 (M þ Na): 431.2270. Found:
431.2268.
Methyl (3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-(4-phen-
ethyl[1,2,3]triazol-1-yl)cyclohex-1-ene-1-carboxylate (26). Com-
pound 26 (370 mg, 76%) was obtained as a pale yellow foam
from compound 9 (350 mg, 1.08 mmol) and 1-phenyl-but-3-yne
(0.2 mL, 1.40 mmol) using the procedure as that described to
obtain 23. 1H NMR (CDCl3, 500 MHz): δ 7.29 (2H, dd, J =7.5
Hz, Ar), 7.21 (1H, dd, J=7.5 Hz, Ar), 7.19 (2H, dd, J = 7.5 Hz,
Ar), 7.24 (1H, s, H-50), 6.74 (1H, br dd, J2,3=J2,6b=2.5 Hz, H-2),
6.02 (1H, dddd, J3,4=10.0, J3,6a=1.5, J3,6b=3.0 Hz, H-3), 5.81
(1H, d, J-NH,4 =7.5 Hz, -NHAc), 4.18 (1H, ddd, J5,4 =10.0,
(C-3), 57.0 (C-4), 52.9 (-COOCH3), 41.95 and 41.92 (2C), 33.0
(C-6), 27.3 and 27.0 (CH3CH2)2CH-O-), 24.62 and 24.61 (2C),
23.0 (-NHCOCH3), 9.9 and 9.8 (CH3CH2)2CH-O-). HRMS
Calcd for C22H35N4O5 (M þ H): 435.2607. Found: 435.2607.
C22H34N4NaO5 (M þ Na): 457.2427. Found: 457.2423.
Methyl (3S,4R,5R)-4-Acetamido-3-[4-((17r)-estra-1,3,5(10)-
triene-3,17-dihydroxy-17-yl)[1,2,3]triazol-1-yl]-5-(1-ethylpropoxy)-
cyclohex-1-ene-1-carboxylate (29). Compound 29 (507 mg, 76%)
was obtained as a pale yellow foam from compound 9 (350 mg,
1.08 mmol) and 17R-ethynylestradiol (383.6 mL, 1.30 mmol)
1
using the procedure as that described to obtain 23. H NMR
(CD3OD, 600 MHz): δ 7.77 (1H, s, H-50), 6.99 (1H, d, J=8.5 Hz,
Ar), 6.78 (1H, br dd, J2,3=2.5, J2,6b=3.0 Hz, H-2), 6.50 (1H, dd,
J=8.5, 3.0 Hz, Ar), 6.46 (1H, d, J=3.0 Hz, Ar), 5.53 (1H, dddd,
J3,4 = 9.5, J3,6a =2.5, J3,6b = 3.0 Hz, H-3), 4.24 (1H, dd, J4,5 =
10.0 Hz, H-4), 3.88 (1H, ddd, J5,6a=5.5, J5,6b=9.5 Hz, H-5), 3.78
(3H, s, -COOCH3), 3.38 (1H, m, (CH3CH2)2CH-O-), 3.02 (1H, br
ddd, J6a,6b = 18.0 Hz, H-6a), 2.81-2.71 (2H, m), 2.46-2.39
(1H, m), 2.40 (1H, dddd, H-6b), 2.17-2.12 (1H, m), 2.10-2.05
(1H, m), 2.0-1.88 (3H, m), 1.84 (3H, s, -NHCOCH3), 1.64-1.59
(2H, m), 1.56-1.45 (5H, m, (CH3CH2)2CH-O-), 1.44-1.27 (3H,
m), 1.03 (3H, s, -CH3), 0.91 and 0.86 (3H, t, J=7.8 Hz, (CH3-
CH2)2CH-O-), 0.66 (1H, ddd). 13C NMR (CD3OD, 150 MHz):
δ 173.4 (-NHCOCH3), 167.6 (-COOCH3), 156.0 (1C, Ar), 155.6
(C-40), 139.0 (1C, Ar), 134.8 (C-2), 132.74 (1C, Ar), 132.73 (C-1),
127.3 (1C, Ar), 123.8 (C-50), 116.2 and 113.8 (2C, Ar), 83.3 (1C),
83.0 (CH3CH2)2CH-O-), 74.3 (C-5), 62.8 (C-3), 57.2 (C-4), 52.9
(-COOCH3), 49.5, 48.5, 44.8, 41.2, 38.7, and 34.4 (6C), 33.3 (C-6),
30.9, 28.8, and 27.7 (3C), 27.3 and 27.0 (CH3CH2)2CH-O-), 24.8
(1C), 23.1 (-NHCOCH3), 15.0 (-CH3), 9.9 and 9.8 (CH3CH2)2-
CH-O-). HRMS Calcd for C35H49N4O6 (M þ H): 621.3652.
Found: 621.3650.
J5,6a=5.5, J5,6b=10.0 Hz, H-5), 3.88 (1H, ddd, H-4), 3.79 (3H, s,
-COOCH3), 3.35 (1H, m, (CH3CH2)2CH-O-), 3.08-2.98 (5H,
m, H-6a, PhCH2CH2-), 2.38 (1H, dddd, J6b,6a=18.0 Hz, H-6b),
1.87 (3H, s, -NHCOCH3), 1.56-1.45 (4H, m, (CH3CH2)2CH-
O-), 0.91 and 0.87 (3H, t, J =7.8 Hz, (CH3CH2)2CH-O-). 13C
NMR (CDCl3, 125 MHz): δ 170.8 (-NHCOCH3), 165.9 (-COO-
CH3), 147.3 (C-40), 141.0, 128.32, 128.30, and 126.0 (6C, Ar),
134.1 (C-2), 131.0 (C-1), 121.0 (C-50), 81.4 (CH3CH2)2CH-O-),
72.5 (C-5), 60.4 (C-3), 55.6 (C-4), 52.1 (-COOCH3), 35.5
(PhCH2CH2-), 31.8 (C-6), 27.6 (PhCH2CH2-), 26.0 and 25.6
(CH3CH2)2CH-O-), 23.1 (-NHCOCH3), 9.4 and 9.2 (CH3CH2)2-
CH-O-). HRMS Calcd for C25H35N4O4 (M þ H): 455.2658.
Found: 455.2659. C25H34N4NaO4 (M þ Na): 477.2478. Found:
477.2473.
Methyl (3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(1-
hydroxycyclohexyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxy-
late (27). Compound 27 (295 mg, 85%) was obtained as a
colorless foam from compound 9 (250 mg, 0.77 mmol) and
1-ethynylcyclohexanol (191.4 mg, 0.92 mmol) using the proce-
(3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(3-hydroxy-
propyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxylic Acid (1).
NaOH (32 mg, 0.8 mmol) was added to a solution of the methyl
ester 23 (65 mg, 0.16 mmol) in a 1:1 mixture of MeOH and water
(3 mL), and the reaction mixture was stirred at room tempera-
ture for 3-4 h. The pH of the reaction mixture was adjusted to
6 using 0.1 N HCl, and solvents were evaporated. The crude
compound was dissolved in MeOH and passed through a pad
of silica gel, and the filtrate was concentrated to give a pale
yellow gum. Addition of ethyl acetate precipitated the desired
1
dure as that described to obtain 23. H NMR (CD3OD, 600
MHz): δ 7.84 (1H, s, H-50), 6.75 (1H, br dd, J2,3=2.5, J2,6b=3.0
Hz, H-2), 5.54 (1H, dddd, J3,4=9.5, J3,6a =1.8, J3,6b =3.5 Hz,
H-3), 4.21 (1H, dd, J4,5 =10.0 Hz, H-4), 3.91 (1H, ddd, J5,6a =
5.5, J5,6b = 9.0 Hz, H-5), 3.77 (3H, s, -COOCH3), 3.40 (1H, m,
(CH3CH2)2CH-O-), 3.01 (1H, ddd, J6a,6b=18.0 Hz, H-6a), 2.42
(1H, dddd, H-6b), 2.04-1.98 (2H, m), 1.84 (3H, s, -NHCO-
CH3), 1.83-1.72 (4H, m), 1.62-1.56 (7H, m, (CH3CH2)2CH-O-
), 1.42-1.35 (1H, m), 0.92 and 0.86 (3H, t, J = 7.8 Hz,
(CH3CH2)2CH-O-). 13C NMR (CD3OD, 150 MHz): δ 173.5
(-NHCOCH3), 167.6 (-COOCH3), 157.2 (C-40), 134.6 (C-2),
132.9 (C-1), 121.9 (C-50), 83.0 (CH3CH2)2CH-O-), 74.1 (C-5),
70.4 (1C), 62.8 (C-3), 57.0 (C-4), 52.9 (-COOCH3), 39.02 and
39.0 (2C), 33.1 (C-6), 27.3 and 27.0 (CH3CH2)2CH-O-), 26.7
(1C), 23.3 and 23.2 (2C), 23.0 (-NHCOCH3), 9.9 and 9.8
(CH3CH2)2CH-O-). HRMS Calcd for C23H37N4O5 (M þ H):
449.2764. Found: 449.2763. C23H36N4NaO5 (M þ Na): 471.2583.
Found: 471.2583.
1
compound 1 as a colorless powder (28 mg, 44%). H NMR
(CD3OD, 600 MHz): δ 7.76 (1H, s, H-50), 6.70 (1H, br t, H-2),
5.51 (1H, br d, J3,4 =9.0 Hz, H-3), 4.19 (1H, dd, J4,5 =9.6 Hz,
H-4), 3.89 (1H, dt, J5,6a =5.4, J5,6b =9.0 Hz, H-5), 3.57 (2H, t,
J = 6.6 Hz, HOCH2CH2CH2-), 3.40 (1H, m, J = 6.0 Hz,
(CH3CH2)2CH-O-), 3.01 (1H, br dd, J6a,6b = 18 Hz, H-6a),
2.76 (2H, t, J=7.8 Hz, HOCH2CH2CH2-), 2.39 (1H, ddt, J6b,2
=
J6b,3 = 3.0 Hz, H-6b), 1.87 (2H, m, J = 7.2 Hz, HOCH2-
CH2CH2-), 1.84 (3H, s, -NHCOCH3), 1.56-1.45 (4H, m, (CH3-
CH2)2CH-O-), 0.92 and 0.86 (3H, t, J = 7.8 Hz, (CH3CH2)2-
CH-O-). 13C NMR (CD3OD, 150 MHz): δ 173.5 (-NHCOCH3),
169.3 (-COOH), 149.2 (C-40), 134.2 (C-1), 133.7 (C-2), 122.6
(C-50), 83.0 (CH3CH2)2CH-O-), 74.3 (C-5), 63.0 (C-3), 62.0
(HOCH2CH2CH2-), 57.1 (C-4), 33.3 (HOCH2CH2CH2-), 33.3
(C-6), 27.3 and 27.0 (CH3CH2)2CH-O-), 23.0 (-NHCOCH3),
22.9 (HOCH2CH2CH2-), 9.9 and 9.8 (CH3-CH2)2-CH-O-).
HRMS Calcd for C19H31N4O5 (M þ H): 395.2294. Found:
395.2282.
(3S,4R,5R)-4-Acetamido-5-(1-ethyl-propoxy)-3-[4-(1-hydroxy-
propyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxylic Acid (2).
Compound 2 (41 mg, colorless powder, 42% yield) was obtained
as a 1:1 mixture of diastereomers from compound 24 (100 mg,
0.25 mmol) using the same procedure as described to obtain 1.
Data for the mixture of diastereomers. 1H NMR (CD3OD, 500
MHz): δ 7.89 and 7.87 (1H, s, H-50), 6.76 and 6.75 (1H, br t,
H-2), 5.50 (2H, br d, J3,4 =9.0 Hz, H-3), 4.73 and 4.72 (1H, dd,
Methyl (3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(1-
hydroxycyclopentyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxy-
late (28). Compound 28 (360 mg, 90%) was obtained as a
colorless foam from compound 9 (300 mg, 0.93 mmol) and
1-ethynylcyclopentanol (0.13 mL, 1.12 mmol) using the proce-
1
dure as that described to obtain 23. H NMR (CD3OD, 600
MHz): δ 7.83 (1H, s, H-50), 6.75 (1H, br dd, J2,3=J2,6b=2.5 Hz,
H-2), 5.55 (1H, dddd, J3,4=9.6, J3,6a=1.8, J3,6b=3.6 Hz, H-3),
4.20 (1H, dd, J4,5 =9.6 Hz, H-4), 3.92 (1H, ddd, J5,6a =5.5 Hz,
J5,6b = 9.6 Hz, H-5), 3.78 (3H, s, -COOCH3), 3.39 (1H, m,
(CH3CH2)2CH-O-), 3.01 (1H, br ddd, J6a,6b = 18.0 Hz, H-6a),
2.42 (1H, dddd, H-6b), 2.11-2.04 (2H, m), 1.99-1.89 (4H, m),
1.84 (3H, s, -NHCOCH3), 1.83-1.76 (2H, m), 1.57-1.44 (4H, m,
(CH3CH2)2CH-O-), 0.92 and 0.86 (3H, t, J = 7.8 Hz, (CH3-
CH2)2CH-O-). 13C NMR (CD3OD, 150 MHz): δ 173.6 (-NH-
COCH3), 167.6 (-COOCH3), 156.1 (C-40), 134.5 (C-2), 133.0 (C-1),
121.9 (C-50), 83.0 (CH3CH2)2CH-O-), 79.7 (1C), 74.8 (C-5), 62.7
J = 7.0 Hz, CH3CH2CH(OH)-), 4.24 and 4.22 (1H, dd, J4,5 =
10.0 Hz, H-4), 3.91 (2H, dt, J5,6a = 5.5, J5,6b = 9.5 Hz, H-5),

7388 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Mohan et al.
3.41 (2H, m, J=5.5 Hz, (CH3CH2)2CH-O-), 3.0 (2H, br dd,
J6a,6b = 18 Hz, H-6a), 2.42 (2H, ddt, J6b,2 = J6b,3 = 3.5 Hz,
H-6b), 1.93-1.81 (4H, m, CH3CH2CH(OH)-), 1.86 and 1.85
(3H, s, -NHCOCH3), 1.59-1.47 (8H, m, (CH3CH2)2CH-O-),
0.96 and 0.95 (3H, t, J = 7.5 Hz, CH3CH2CH(OH)-), 0.94 and
0.88 (6H, t, J=7.5 Hz, (CH3CH2)2CH-O-). 13C NMR (CD3OD,
125 MHz): δ 172.2 and 172.1 (-NHCOCH3), 167.5 (-COOH),
151.9 and 151.8 (C-40), 132.9 (C-2), 132.4 and 132.3 (C-1), 121.3
and 121.2 (C-50), 81.7 (CH3CH2)2CH-O-), 73.0 (C-5), 67.9 and
67.8 (CH3CH2CH(OH)-), 61.8 and 61.7 (C-3), 55.8 and 55.7
(C-4), 31.9 and 31.8 (C-6), 30.2 and 30.1 (CH3CH2CH(OH)-),
26.0 and 25.7 (CH3CH2)2CH-O-), 21.7 (-NHCOCH3), 9.0 and
8.9 (CH3CH2CH(OH)-), 8.7, 8.6, and 8.5 (CH3-CH2)2-CH-O-).
HRMS Calcd for C19H31N4O5 (M þ H): 395.2294. Found:
395.2284.
(CH3CH2)2CH-O-), 2.24 (1H, dd, J6a,6b =14.0 Hz, H-6a), 1.96
(1H, ddd, H-6b), 1.87 (3H, s, -NHCOCH3), 1.59 (6H, s,
(CH3)2C(OH)-), 1.60-1.45 (4H, m, (CH3CH2)2CH-O-), 0.93
and 0.89 (3H, t, J=7.8 Hz, (CH3CH2)2CH-O-). HRMS Calcd
for C19H30DN4O5 (M þ H): 396.2356. Found: 396.2356. C19-
H29DN4NaO5 (M þ Na): 418.2176. Found: 418.2176. C19H29-
DKN4O5 (M þ K): 434.1915. Found: 434.1912.
Data for 36 (from a Mixture Containing 28% of 32). 1H NMR
(CD3OD, 600 MHz): δ 7.80 (1H, s, H-50), 6.98 (1H, br s, H-2),
5.80 (1H, ddd, J3,4 = 5.5, J3,2 = 2.5, J3,6a = 3.0 Hz, H-3), 4.65
(1H, dd, J4,5 =5.5 Hz, H-4), 3.92 (1H, ddd, J5,6a =3.0, J5,6b
=
3.5 Hz, H-5), 3.40 (1H, m, J=6.0 Hz, (CH3CH2)2CH-O-), 2.69
(1H, dddd, J6a,6b=19.0, J6a,2=2.5 Hz, H-6a), 2.60 (1H, br ddd-
like, H-6b), 1.79 (3H, s, -NHCOCH3), 1.58 (6H, s, (CH3)2-
C(OH)-), 1.61-1.45 (4H, m, (CH3CH2)2CH-O-), 0.98 and 0.90
(3H, t, J=7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CD3OD, 150
MHz): δ 172.1 (-NHCOCH3), 167.9 (-COOH), 155.6 (C-40),
132.3 (C-1), 131.3 (C-2), 120.6 (C-50), 80.8 (CH3CH2)2CH-O-),
71.5 (C-5), 67.7 ((CH3)2C(OH)-), 56.8 (C-3), 49.9 (C-4), 29.3 and
29.2 ((CH3)2C(OH)-), 26.8 (C-6), 26.1 and 26.0 (CH3CH2)2CH-O-),
20.9 (-NHCOCH3), 8.7 and 8.5 (CH3CH2)2CH-O-).
(3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(1-hydroxy-
1-methylethyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxylic
Acid (3). Compound 3 (78 mg, colorless powder, 40% yield) was
obtained from compound 25 (200 mg, 0.49 mmol) using the
same procedure as described to obtain 1. From the ethyl acetate
soluble layer, compound 32 (80 mg, colorless powder, 41%
yield) was obtained by crystallization. Chromatographic puri-
fication of the mother liquor yielded compound 36 as a mixture
containing 28% of 32 (25 mg, 13% yield). Similarly, the corre-
sponding deuterated compounds 3(D), 32(D), and 36(D) were
obtained from 25 using deuterated sodium hydroxide and deu-
terated methanol.
Data for 36(D) (from a Mixture Containing 33% of 32(D)). 1H
NMR (CD3OD, 600 MHz): δ 7.81 (1H, s, H-50), 6.99 (1H, br s,
H-2), 4.64 (1H, d, J4,5=5.5 Hz, H-4), 3.93 (1H, ddd-like, J5,6a=
3.5, J5,6b =3.0 Hz, H-5), 3.44 (1H, m, J=6.0 Hz, (CH3CH2)2-
CH-O-), 2.69 (1H, ddd, J6a,6b=19.2, J6a,2=2.5 Hz, H-6a), 2.60
(1H, br d, H-6b), 1.79 (3H, s, -NHCOCH3), 1.56 (6H, s,
(CH3)2C(OH)-), 1.61-1.45 (4H, m, (CH3CH2)2CH-O-), 0.98
and 0.90 (3H, t, J =7.8 Hz, (CH3CH2)2CH-O-).
1
Data for 3. H NMR (CD3OD, 600 MHz): δ 7.83 (1H, s,
H-50), 6.74 (1H, br t, H-2), 5.53 (1H, br d, J3,4 = 8.4 Hz, H-3),
(3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-(4-phenethyl-
[1,2,3]triazol-1-yl)cyclohex-1-ene-1-carboxylic Acid (4). Com-
pound 4 (46 mg, colorless powder, 32% yield) was obtained
from compound 26 (150 mg, 0.37 mmol) using the same pro-
cedure as described to obtain 1. From the ethyl acetate soluble
layer, compound 33 (44 mg, colorless powder, 30% yield) was
obtained by crystallization.
4.20 (1H, dd, J4,5=9.6 Hz, H-4), 3.90 (1H, dt, J5,6a=5.4, J5,6b
9.0 Hz, H-5), 3.39 (1H, m, J = 5.4 Hz, (CH3CH2)2CH-O-), 3.0
(1H, br dd, J6a,6b =18 Hz, H-6a), 2.41 (1H, ddt, J6b,2 =J6b,3
=
=
3.0 Hz, H-6b), 1.84 (3H, s, -NHCOCH3), 1.57 (6H, s, (CH3)2-
C(OH)-), 1.60-1.46 (4H, m, (CH3CH2)2CH-O-), 0.92 and 0.86
(3H, t, J=7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CD3OD, 150
MHz): δ 173.6 (-NHCOCH3), 168.9 (-COOH), 157.5 (C-40),
134.1 (C-2), 133.7 (C-1), 121.2 (C-50), 83.0 (CH3CH2)2CH-O-),
74.2 (C-5), 69.2 ((CH3)2C(OH)-), 62.9 (C-3), 57.1 (C-4), 33.0
(C-6), 30.7 ((CH3)2C(OH)-), 27.3 and 27.0 (CH3CH2)2CH-O-),
23.0 (-NHCOCH3), 9.9 and 9.8 (CH3CH2)2CH-O-). HRMS
Calcd for C19H31N4O5 (M þ H): 395.2294. Found: 395.2304.
Data for 3(D). 1H NMR (CD3OD, 600 MHz): δ 7.83 (1H, s,
H-50), 6.36 (1H, d, J2,6b = 2.5 Hz, H-2), 4.17 (1H, d, J4,5 = 9.6
Hz, H-4), 3.83 (1H, ddd, J5,6a=5.5 Hz, J5,6b=9.6 Hz, H-5), 3.39
1
Data for 4. H NMR (CD3OD, 600 MHz): δ 7.62 (1H, s,
H-50), 7.29-7.14 (5H, m, Ar), 6.66 (1H, br t, H-2), 5.50 (1H, br d,
J
3,4 =9.6 Hz, H-3), 4.18 (1H, dd, J4,5 =9.6 Hz, H-4), 3.87 (1H,
dt, J5,6a = 5.4, J5,6b = 9.6 Hz, H-5), 3.38 (1H, m, J = 6.0 Hz,
(CH3CH2)2CH-O-), 3.03-2.89 (5H, m, H-6a, PhCH2CH2-), 2.38
(1H, ddt, J6b,6a=18.0, J6b,2=J6b,3=3.0 Hz, H-6b), 1.83 (3H, s,
-NHCOCH3), 1.55-1.45 (4H, m, (CH3CH2)2CH-O-), 0.91 and
0.86 (3H, t, J=7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CD3OD,
150 MHz): δ 173.5 (-NHCOCH3), 168.8 (-COOH), 148.9 (C-40),
142.50, 129.7, 129.6, and 127.3 (Ar), 134.3 (C-2), 133.6 (C-1),
122.7 (C-50), 83.0 (CH3CH2)2CH-O-), 74.3 (C-5), 62.8 (C-3),
57.0 (C-4), 36.7 (PhCH2CH2-), 33.1 (C-6), 28.6 (PhCH2CH2-),
27.3 and 27.0 (CH3CH2)2CH-O-), 23.0 (-NHCOCH3), 9.9 and
9.8 (CH3CH2)2CH-O-). HRMS Calcd for C24H33N4O4 (M þ
H): 441.2502. Found: 441.2511.
(1H, m, J = 6.0 Hz, (CH3CH2)2CH-O-), 3.04 (1H, dd, J6a,6b
=
18.0 Hz, H-6a), 2.41 (1H, ddd, H-6b), 1.84 (3H, s, -NHCOCH3),
1.56 (6H, s, (CH3)2C(OH)-), 1.58-1.45 (4H, m, (CH3CH2)2CH-
O-), 0.92 and 0.86 (3H, t, J = 7.8 Hz, (CH3CH2)2CH-O-).
HRMS Calcd for C19H30DN4O5 (M þ H): 396.2356. Found:
396.2356. C19H29DN4NaO5 (M þ Na): 418.2176. Found:
418.2176. C19H219DKN4O5 (M þ K): 434.1915. Found: 434.1923.
Data for 32. H NMR (CD3OD, 500 MHz): δ 7.96 (1H, s,
H-50), 6.73 (1H, d, J2,1=2.5 Hz, H-2), 5.13 (1H, br dd-like, H-4),
3.86 (1H, ddd, J5,4 =2.5, J5,6a = 5.0, J5,6b = 2.0 Hz, H-5), 3.61
(1H, dddd, J1,6a=5.0, J1,6b=11.0, J1,4=2.5 Hz, H-1), 3.54 (1H,
m, J=6.0 Hz, (CH3CH2)2CH-O-), 2.25 (1H, ddd, J6a,6b =14.0
Hz, H-6a), 1.96 (1H, ddd, H-6b), 1.87 (3H, s, -NHCOCH3), 1.59
(6H, s, (CH3)2C(OH)-), 1.60-1.44 (4H, m, (CH3CH2)2CH-O-),
0.94 and 0.89 (3H, t, J = 7.8 Hz, (CH3CH2)2CH-O-). 13C NMR
(CD3OD, 125 MHz): δ 176.4 (-COOH), 173.0 (-NHCOCH3),
157.2 (C-40), 134.1 (C-3), 122.3 (C-2), 119.7 (C-50), 82.0 (CH3-
CH2)2CH-O-), 74.8 (C-5), 69.1 ((CH3)2C(OH)-), 49.0 (C-4), 38.8
(C-1), 30.72 and 30.70 ((CH3)2C(OH)-), 27.8 and 27.7 (CH3-
CH2)2CH-O-), 27.4 (C-6), 22.5 (-NHCOCH3), 10.3 and 10.2 (CH3-
CH2)2CH-O-). HRMS Calcd for C19H31N4O5 (M þ H): 395.2294.
Found: 395.2295. C19H30N4NaO5 (M þ Na): 417.2114. Found:
417.2119. C19H30KN4O5 (M þ K): 433.1853. Found: 433.1855.
Data for 32(D). 1H NMR (CD3OD, 600 MHz): δ 7.97 (1H, s,
H-50), 6.72 (1H, s, H-2), 5.13 (1H, d, J4,5 = 2.5 Hz, H-4), 3.86
(1H, ddd, J5,6a=4.5, J5,6b=2.0 Hz, H-5), 3.54 (1H, m, J=6.0 Hz,
1
Data for 33. H NMR (CD3OD, 500 MHz): δ 7.73 (1H, s,
H-50), 7.26-7.14 (5H, m, Ar), 6.68 (1H, br d, J2,1=2.5 Hz, H-2),
5.07 (1H, br dd-like, H-4), 3.83 (1H, ddd, J5,4 =2.5, J5,6a =4.5,
J5,6b = 2.0 Hz, H-5), 3.59 (1H, dddd, J1,6a = 5.0, J1,6b = 11.0,
J1,4=3.0 Hz, H-1), 3.52 (1H, m, J=6.0 Hz, (CH3CH2)2CH-O-),
3.04-2.96 (4H, m, Ph-CH2-CH2-), 2.24 (1H, ddd, J6a,6b = 13.5
Hz, H-6a), 1.94 (1H, ddd, H-6b), 1.85 (3H, s, -NHCOCH3),
1.60-1.43 (4H, m, (CH3CH2)2CH-O-), 0.93 and 0.87 (3H, t, J=
7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CD3OD, 125 MHz): δ
176.4 (-COOH), 173.0 (-NHCOCH3), 148.5 (C-40), 142.3, 129.6,
127.3 (Ar), 133.9 (C-3), 122.1 (C-2), 121.3 (C-50), 82.1 (CH3-
CH2)2CH-O-), 74.8 (C-5), 48.8 (C-4), 38.8 (C-1), 36.5 (PhCH2-
CH2-), 28.3 (PhCH2CH2-), 27.8 and 27.7 (CH3CH2)2CH-O-),
27.4(C-6), 22.5(-NHCOCH3), 10.3 and 10.2 (CH3CH2)2CH-O-).
HRMS Calcd for C24H33N4O4 (M þ H): 441.2502. Found:
441.2499. C24H32N4NaO4 (M þ Na): 463.2321. Found: 463.2318.
(3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(1-hydroxy-
cyclohexyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxylic Acid (5).
A mixture of compound 27 (137 mg, 0.31 mmol) and trimethyltin

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7389
hydroxide (442 mg, 2.45 mmol) in 8 mL of dichloroethane
(DCE) was heated at 80 °C for 5 h. Solvents were removed
under reduced pressure, and the crude mass was purified by
column chromatography (EtOAc/MeOH, 7:3 (v/v)). Compound
5 was obtained as a colorless foam (80 mg, 61%). H NMR
(CD3OD, 600 MHz): δ 7.85 (1H, s, H-50), 6.71 (1H, br t, H-2),
(56 mg, 58%). 1H NMR (CD3OD, 600 MHz): δ 6.67 (1H, ddd,
J2,3=J2,6b=3.0, J2,6a=0.6 Hz, H-2), 4.21 (1H, dddd, J3,4=9.0,
J
3,6a=1.8, J3,6b=3.6 Hz, H-3), 3.93 (1H, dd, J4,5=9.6 Hz, H-4),
3.68 (1H, ddd, J5,6a = 5.4, J5,6b = 9.0 Hz, H-5), 3.34 (1H, m,
(CH3CH2)2CH-O-), 2.85 (1H, dddd, J6a,6b = 18.0 Hz, H-6a),
2.23 (1H, dddd, H-6b), 1.99 (3H, s, -NHCOCH3), 1.53-1.44
(4H, m, (CH3CH2)2CH-O-), 0.90 and 0.89 (3H, t, J = 7.8 Hz,
(CH3CH2)2CH-O-). 13C NMR (CD3OD, 150 MHz): δ 173.9
(-NHCOCH3), 169.0 (-COOH), 135.3 (C-2), 132.6 (C-1), 83.0
(CH3CH2)2CH-O-), 74.1 (C-5), 62.8 (C-3), 56.4 (C-4), 32.7 (C-6),
27.3 and 27.0 (CH3CH2)2CH-O-), 23.3 (-NHCOCH3), 10.0 and
9.8 (CH3CH2)2CH-O-). HRMS Calcd for C14H23N4O4 (M þ H):
311.1719. Found:311.1716. C14H22N4NaO4 (M þ Na):333.1539.
Found: 333.1535. C14H22KN4O4 (M þ K): 349.1278. Found:
349.1288.
(3S,4R,5R)-4-Acetamido-3-amino-5-(1-ethylpropoxy)cyclohex-
1-ene-1-carboxylic Acid(38). Hydrogen gas was bubbled through
a mixture of compound 40 (20 mg, 0.06 mmol) and Lindlar’s
catalyst (8 mg) in 4 mL of ethanol at room temperature for 2.5 h.
The reaction mixture was filtered through Celite, and the fil-
trate was concentrated and purified by column chromatography
(EtOAc/MeOH/H2O, 2:3:0.1 (v/v)). Compound 38 was ob-
tained as yellow foam (14 mg, 82% yield). 1H NMR (D2O,
1
5.52 (1H, br d, J3,4 = 9.0 Hz, H-3), 4.22 (1H, dd, J4,5
9.6 Hz, H-4), 3.89 (1H, dt, J5,6a= 6.0, J5,6b= 9.6 Hz, H-5), 3.40
(1H, m, J=5.4 Hz, (CH3CH2)2CH-O-), 3.01 (1H, br dd, J6a,6b
=
=
18 Hz, H-6a), 2.41 (1H, ddt, J6b,2 = J6b,3 = 3.5 Hz, H-6b),
2.04-1.98 (2H, m), 1.84 (3H, s, -NHCOCH3), 1.83-1.72 (4H,
m), 1.61-1.56 (7H, m, (CH3CH2)2CH-O-), 1.42-1.36 (1H, m),
0.92 and 0.86 (3H, t, J=7.8 Hz, (CH3CH2)2CH-O-). 13C NMR
(CD3OD, 150 MHz): δ 173.5 (-NHCOCH3), 169.4 (-COOH),
157.1 (C-40), 134.4 (C-1), 133.5 (C-2), 121.8 (C-50), 83.0 (CH3-
CH2)2CH-O-), 74.3 (C-5), 70.4 (1C), 63.0 (C-3), 57.1 (C-4), 39.0
and 38.9 (2C), 33.2 (C-6), 27.3 and 27.0 (CH3CH2)2CH-O-), 26.7
(1C), 23.3 and 23.2 (2C), 23.0 (-NHCOCH3), 9.9 and 9.8
(CH3CH2)2CH-O-). HRMS Calcd for C22H35N4O5 (M þ H):
435.2607. Found: 435.2611.
(3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-[4-(1-hydroxy-
cyclopentyl)[1,2,3]triazol-1-yl]cyclohex-1-ene-1-carboxylic Acid (6).
Compound 6 (63 mg, 62%, colorless foam) was obtained from
compound 28 (106 mg, 0.24 mmol) using the same procedure as
600 MHz): δ 6.29 (1H, br t, H-2), 4.07 (1H, dd, J4,3 = J4,5
9.6 Hz, H-4), 4.0 (1H, br d, H-3), 3.80 (1H, dt, J5,6a=5.4, J5,6b
=
=
1
described to obtain 5. H NMR (CD3OD, 600 MHz): δ 7.84
(1H, s, H-50), 6.74 (1H, br t, H-2), 5.54 (1H, br d, J3,4 =9.0 Hz,
H-3), 4.20 (1H, dd, J4,5=9.6 Hz, H-4), 3.90 (1H, dt, J5,6a=5.4,
9.6 Hz, H-5), 3.48 (1H, m, J=5.4 Hz, (CH3CH2)2CH-O-), 2.89
(1H, br dd, J6a,6b=17.4 Hz, H-6a), 2.33 (1H, ddt, J6b,2=J6b,3
=
J
5,6b=9.6 Hz, H-5), 3.39 (1H, m, J=5.4 Hz, (CH3CH2)2CH-O-),
2.99 (1H, br dd, J6a,6b = 18 Hz, H-6a), 2.41 (1H, ddt, J6b,2
6b,3 = 3.5 Hz, H-6b), 2.12-2.04 (2H, m), 1.98-1.89 (4H, m),
3.0 Hz, H-6b), 2.06 (3H, s, -NHCOCH3), 1.59-1.44 (4H, m,
(CH3CH2)2CH-O-), 0.86 (6H, t, J=7.8 Hz, (CH3CH2)2CH-O-).
13C NMR (D2O, 150 MHz): δ 174.9 (-NHCOCH3), 173.8
(-COOH), 138.7 (C-1), 125.0 (C-2), 83.3 (CH3CH2)2CH-O-),
73.4 (C-5), 52.7 (C-4), 52.6 (C-3), 32.3 (C-6), 25.3 (CH3CH2)2-
CH-O-), 22.3 (-NHCOCH3), 8.7 and 8.4 (CH3CH2)2CH-O-).
HRMS Calcd for C14H25N2O4 (M þ H): 285.1814. Found:
285.1813.
=
J
1.84 (3H, s, -NHCOCH3), 1.82-1.77 (2H, m), 1.57-1.45 (4H,
m, (CH3CH2)2CH-O-), 0.92 and 0.86 (3H, t, J = 7.8 Hz,
(CH3CH2)2CH-O-). 13C NMR (CD3OD, 150 MHz): δ 173.5
(-NHCOCH3), 172.9 (-COOH), 156.0 (C-40), 138.6 (C-1), 129.4
(C-2), 121.5 (C-50), 82.9 (CH3CH2)2CH-O-), 79.6 (1C), 74.7 (C-
5), 63.1 (C-3), 57.3 (C-4), 41.9 and 41.8 (2C), 34.0 (C-6), 27.4 and
27.0 (CH3CH2)2CH-O-), 24.6 (2C), 23.0 (-NHCOCH3), 10.0
and 9.8 (CH3CH2)2CH-O-). HRMS Calcd for C21H33N4O5
(M þ H): 421.2451. Found: 421.2454.
Methyl (3S,4R,5R)-4-Acetamido-3-amino-5-(1-ethylpropoxy)-
cyclohex-1-ene-1-carboxylate (41). Hydrogen gas was bubbled
through a stirred mixture of compound 9 (250 mg, 0.77 mmol)
and Lindlar’s catalyst (80 mg) in 5 mL of EtOH at room
temperature for 3 h. The reaction mixture was then filtered
through a pad of Celite, and the filtrate was concentrated to
dryness. The crude material was purified by column chromato-
graphy (MeOH/EtOAc 1:2) to give compound 41 (140 mg, 61%)
as a pale yellow oil. 1H NMR (CD3OD, 600 MHz): δ 6.78 (1H,
br ddd-like, H-2), 3.82 (1H, dd, J4,3 = 7.8, J4,5 = 9.0 Hz, H-4),
3.75 (3H, s, -COOCH3), 3.67 (1H, ddd, J5,6a = 5.4, J5,6b = 7.8
Hz, H-5), 3.41 (1H, br ddd-like, H-3), 3.33 (1H, m, (CH3CH2)2-
CH-O-), 2.73 (1H, dddd, J6a,6b=18.0, J6a,3=1.8, J6a,2=1.2 Hz,
H-6a), 2.31 (1H, dddd, J6b,3 =J6b,2 =3.0 Hz, H-6b), 2.0 (3H, s,
-NHCOCH3), 1.54-1.41 (4H, m, (CH3CH2)2CH-O-), 0.90 and
0.88 (3H, t, J=7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (CD3OD,
150 MHz): δ 174.1 (-NHCOCH3), 168.6 (-COOCH3), 140.7 (C-2),
129.3 (C-1), 82.7 (CH3CH2)2CH-O-), 74.5 (C-5), 57.6 (C-4), 53.5
(C-3), 52.6 (-COOCH3), 31.5 (C-6), 27.4 and 27.1 (CH3-
CH2)2CH-O-), 23.2 (-NHCOCH3), 10.0 and 9.8 (CH3CH2)2-
CH-O-). HRMS Calcd for C15H27N2O4 (M þ H): 299.1965.
Found: 299.1963.
(3S,4R,5R)-4-Acetamido-3-[4-((17r)-estra-1,3,5(10)-triene-
3,17-dihydroxy-17-yl)[1,2,3]triazol-1-yl]-5-(1-ethylpropoxy)cyclo-
hex-1-ene-1-carboxylic Acid (7). Compound 7 (79 mg, 72%,
yellow foam) was obtained from compound 29 (115 mg, 0.18
1
mmol) using the same procedure as described to obtain 5. H
NMR (CD3OD, 600 MHz): δ 7.78 (1H, s, H-50), 6.99 (1H, d, J=
8.4 Hz, Ar), 6.78 (1H, br t, H-2), 6.50 (1H,br dd, J=8.4 Hz, Ar),
6.45 (1H, d, J=2.4 Hz, Ar), 5.53 (1H, br d, J3,4=9.6 Hz, H-3),
4.26 (1H, dd, J4,5 = 10.0 Hz, H-4), 3.87 (1H, dt, J5,6a = 5.4,
J5,6b=9.6 Hz, H-5), 3.38 (1H, m, J=6.0 Hz, (CH3CH2)2CH-O-),
3.01 (1H, br dd, J6a,6b = 18.0 Hz, H-6a), 2.80-2.70 (2H, m),
2.46-2.41 (1H, m), 2.40 (1H, ddt, J6b,2 =J6b,3 =3.5 Hz, H-6b),
2.16-1.88 (5H, m), 1.85 (3H, s, -NHCOCH3), 1.64-1.59 (2H,
m), 1.56-1.45 (5H, m, (CH3CH2)2CH-O-), 1.44-1.27 (3H, m),
1.03 (3H, s), 0.91 and 0.86 (3H, t, J=7.8 Hz, (CH3CH2)2CH-O-),
0.66 (1H, m). 13C NMR (CD3OD, 150 MHz): δ 173.3 (-NH-
COCH3), 168.8 (-COOH), 156.0 (1C, Ar), 155.6 (C-40), 139.0
(1C, Ar), 134.6 (C-2), 133.3 (C-1), 132.7 and 127.3 (2C, Ar),
123.9 (C-50), 116.2 and113.8 (2C, Ar), 83.3 (CH3CH2)2CH-O-),
83.0 (1C), 74.4 (C-5), 62.9 (C-3), 57.2 (C-4), 49.5, 48.5, 44.8, 41.2,
38.6, and 34.4 (6C), 33.4 (C-6), 30.9, 28.8, and 27.7 (3C), 27.3
and 27.0 (CH3CH2)2CH-O), 24.8 (1C), 23.2 (-NHCOCH3), 15.0
(1C), 9.9 and 9.8 (CH3CH2)2CH-O-). HRMS Calcd for
C34H47N4O6 (M þ H): 607.3496. Found: 607.3516.
Methyl (3S,4R,5R)-4-Acetamido-3-[2,3-bis(tert-butoxycarbo-
nyl)guanidino]-5-(1-ethylpropoxy)cyclohex-1-ene-1-carboxylate
(42). To a stirred solution of compound 41 (100 mg, 0.34 mmol),
N,N0-bis(tert-butoxycarbonyl)thiourea (110 mg, 0.41 mmol),
and Et3N (15 mL) in dry DMF (5 mL) at 0 °C was added HgCl2
(100 mg, 0.36 mmol). After the addition, the reaction tempera-
ture was maintained at 0 °C for 1 h and then continued at room
temperature for 30 min. The reaction mixture was then diluted
with ethyl acetate and filtered. The filtrate was concentrated
to dryness and purified by column chromatography (MeOH/
CHCl3 1:9) to give compound 42 (102 mg, 56%) as a color-
(3S,4R,5R)-4-Acetamido-3-azido-5-(1-ethylpropoxy)cyclohex-
1-ene-1-carboxylic Acid (40). A mixture of compound 9 (100 mg,
0.31 mmol) and trimethyltin hydroxide (280 mg, 1.54 mmol) in
3 mL of dichloroethane (DCE) was heated at 80 °C for 3.5 h.
Solvents were removed under reduced pressure, and the crude
mass was purified by column chromatography (EtOAc/MeOH,
7:3 (v/v)). Compound 40 was obtained as a colorless foam
less foam. 1H NMR (CD3OD, 600 MHz): δ 6.81 (1H, ddd, J2,3
4.2, J2,6a = J2,6b = 1.8 Hz, H-2), 4.82 (1H, dddd, J3,4 = 4.2,
=

7390 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Mohan et al.
J3,6a = J3,6b = 1.8 Hz, H-3), 4.12 (1H, dd, J4,5 = 6.0 Hz, H-4),
3.88 (1H, ddd, J5,6a = J5,6b = 4.2 Hz, H-5), 3.77 (3H, s,
-COOCH3), 3.29 (1H, m, (CH3CH2)2CH-O-), 2.58 (1H, dddd,
Acknowledgment. We are grateful to Simon Fraser Uni-
versity for financial support in the form of a Prototype
Development Grant and M.v.I. gratefully acknowledges the
financial support of the Honda Foundation, Australia.
J
6a,6b = 18.6 Hz, H-6a), 2.48 (1H, dddd, H-6b), 1.94 (3H, s,
-NHCOCH3), 1.62-1.43 (4H, m, (CH3CH2)2CH-O-), 1.51
and 1.48 (9H, s, -(Boc)2), 0.91 and 0.83 (3H, t, J = 7.8 Hz,
(CH3CH2)2CH-O-). 13C NMR (CD3OD, 150 MHz): δ 173.3
(-NHCOCH3), 168.6 (-COOCH3), 164.6 and 153.7 ([-COOC-
(CH3)3]2), 156.8 ((-NH)(NHBoc)C=NBoc), 135.7 (C-2), 130.4
(C-1), 84.5 and 80.6 ([-COOC(CH3)3]2), 83.1 (CH3CH2)2CH-O-),
72.8 (C-5), 52.68 (-COOCH3)*, 52.67 (C-4)*, 49.8 (C-3), 28.7
and 28.4 ([-COOC(CH3)3]2), 27.9 (C-6), 27.48 and 27.46 (CH3-
CH2)2CH-O-), 22.8 (-NHCOCH3), 10.7 and 9.8 (CH3CH2)2-
CH-O-), where / indicates interchangeable. HRMS Calcd for
C26H45N4O8 (M þ H): 541.3237. Found: 541.3225.
Supporting Information Available: 1H and 13C NMR spectra
of all new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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(3S,4R,5R)-4-Acetamido-5-(1-ethylpropoxy)-3-guanidinocyclo-
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δ 6.70 (1H, dd, J2,3=J2,6a=2.4 Hz, H-2), 4.38 (1H, br d, J3,4
=
9.0 Hz, H-3), 4.07 (1H, dd, J4,5 = 9.0 Hz, H-4), 3.85 (1H, dt,
J5,6a = 5.4, J5,6b = 9.0 Hz, H-5), 3.50 (1H, m, J = 5.4 Hz,
(CH3CH2)2CH-O-), 2.95 (1H, br dd, J6a,6b=18 Hz, H-6a), 2.33
(1H, ddt, J6b,2 = J6b,3 = 3.5 Hz, H-6b), 2.03 (3H, s, -NHCO-
CH3), 1.60-1.43 (4H, m, (CH3CH2)2CH-O-), 0.87 and 0.86
(3H, t, J = 7.8 Hz, (CH3CH2)2CH-O-). 13C NMR (D2O, 150
MHz): δ 174.6 (-NHCOCH3), 169.3 (-COOH), 162.9 (q, JC,F
=
35.3 Hz, CF3COO-), 156.8 (NH2C(NH)-NH-), 135.2 (C-2),
130.5 (C-1), 116.3 (q, JC,F = 290.0 Hz, CF3COO-), 83.4 (CH3-
CH2)2CH-O-), 73.1 (C-5), 54.4 (C-4), 53.6 (C-3), 31.2 (C-6), 25.4
and 25.3 (CH3CH2)2CH-O-), 22.1 (-NHCOCH3), 8.7 and 8.2
(CH3CH2)2CH-O-). HRMS Calcd for C17H27F3N4O6 (M -
CF3COO-): 327.2032. Found: 327.2031.
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[1,2,3]triazol-1-yl)cyclohex-1-ene-1-carboxylic Acid L-Arginine
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(3S,4R,5R)-4-Acetamido-3-[4-((17r)-estra-1,3,5(10)-triene-
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at room temperature for 4 h. The solvents were removed, and the
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