Antibacterial nicotinamide adenine dinucleotide synthetase inhibitors: Amide- and ether-linked tethered dimers with α-amino acid end groups

Article abstract of DOI:10.1021/jm061349l

Tethered dimers incorporating natural α-amino acid end groups were synthesized, including examples in which the previously reported esterase-sensitive ester linker was replaced with more stable amide or ether linkers. These compounds remained effective both as inhibitors of NAD synthetase and as potent antibacterial agents for Gram-positive strains. Studies on nonspecific effects, including detergent properties and promiscuous inhibition, suggested little contribution to observed activities.

Full text of DOI:10.1021/jm061349l

2612
J. Med. Chem. 2007, 50, 2612-2621
Articles
Antibacterial Nicotinamide Adenine Dinucleotide Synthetase Inhibitors: Amide- and
Ether-Linked Tethered Dimers with r-Amino Acid End Groups
Sadanandan E. Velu,†,| Liyuan Mou,†,| Chi-Hao Luan,‡,| Zhengrong W. Yang,| Lawrence J. DeLucas,§,|
Christie G. Brouillette,‡,| and Wayne J. Brouillette*,†,|
Department of Chemistry, Department of Vision Sciences, Department of Optometry, and Center for Biophysical Sciences and Engineering,
UniVersity of Alabama at Birmingham, 901 14th Street South, Birmingham, Alabama 35294
ReceiVed NoVember 21, 2006
Tethered dimers incorporating natural R-amino acid end groups were synthesized, including examples in
which the previously reported esterase-sensitive ester linker was replaced with more stable amide or ether
linkers. These compounds remained effective both as inhibitors of NAD synthetase and as potent antibacterial
agents for Gram-positive strains. Studies on nonspecific effects, including detergent properties and
promiscuous inhibition, suggested little contribution to observed activities.
Introduction
inhibition may thus block both spore germination/outgrowth and
vegetative growth for the spore-forming Bacillus anthracis.
We utilized the protein crystal structure of Bacillus subtilis
NAD synthetase in complex with the natural substrates to guide
the initial design of potential inhibitors.11 As a first approach,
we targeted the NaAD binding subsite with the goal of obtaining
inhibitors that were chemically simpler than NaAD. We, thus,
based early libraries on simple tethered dimers containing
aromatic end groups and a polymethylene linker of varying
length. This approach12,13 resulted in low micromolar inhibitors
of B. subtilis NAD synthetase that also exhibited potent
antibacterial actions. These early tethered dimers contained a
substituted indole at one end and an aromatic group containing
a positively charged N at the other end connected by a
polymethylene linker. SAR studies incorporating substituted
phenols in the place of indole also yielded active compounds,14
and the best example contained a 4-benzyloxy-substituted phenol
at one end and a N,N,N-trimethylammonium phenyl acetate
group at the other end of an 8-carbon polymethylene linker (1,
Figure 1). To further define SAR, here we describe studies
involving substitutions at (1) the ammonium end and (2) the
esterase-sensitive ester linker. Further, we evaluate possible
nonspecific enzyme inhibition actions of the tethered dimers,
including detergent properties and promiscuous behavior.
The need for development of new antibacterial agents has
become a priority of great significance. One factor receiving
major attention is the potential for use of infectious bacteria as
weapons in biowarfare or bioterrorism,1-4 including those
engineered to be resistant against existing drugs. And the natural
emergence of antibiotic-resistant bacteria, due in part to continu-
ing overuse of antibiotics, continues to increase5,6 such that the
majority of Staphylococcus aureus strains are now resistant to
methicillin or other antibiotics. Further, Enterococci have
developed alarming resistance to vancomycin, once considered
an antibiotic of “last resort”. In spite of these threats, the
introduction of new antibacterial drugs for clinical use has
dramatically decreased.
In an effort to identify new antibacterial compounds that act
against novel targets, we have pursued inhibitors of the enzyme
nicotinamide adenine dinucleotide (NADa) synthetase. NAD
synthetase belongs to the amidotransferase family7 and catalyzes
the last step in the biosynthesis of NAD, transforming nicotinic
acid adenine dinucleotide (NaAD) into the amide product NAD
via a two-step process8 involving mixed anhydride derivatization
of the carboxylate, using ATP, followed by reaction with
ammonia (in procaryotes) to provide the amide. The inhibition
of bacterial NAD synthetase appeared likely to provide anti-
bacterial actions since NAD plays an important role in energy
metabolism and numerous biochemical transformations such as
DNA repair, DNA recombination, and protein-ADP ribosyla-
tion.9 Additionally, this enzyme is required for the outgrowth
of spore-forming bacteria into the vegetative cell,10 and its
Results and Discussion
Substitutions at the Ammonium End. (a) R-Amino Acids.
We first proposed to incorporate several N,N,N-trimethylam-
monium derivatives of natural amino acids in place of the
N,N,N-trimethylammonium phenyl acetate end group of com-
pound 1. Because previous SAR studies revealed that the
presence of an aromatic ring near the ammonium group is
favorable, we selected natural R-amino acids containing an
aromatic group, namely, phenylalanine, tyrosine, and tryptophan
(targets 2a-2c in Figure 1).
The syntheses of 2a-2c are summarized in Scheme 1. As
shown, the anion of commercially available 4-benzyloxyphenol
3 was alkylated with 8-bromo-1-octanol to afford alcohol 4.
Intermediate 4 was esterified with the appropriate Boc-protected
amino acids in the presence of EDAC to give the esters 5a-c.
* To whom correspondence should be addressed. Phone: (205) 934-
8288. Fax: (205) 934-2543. E-mail: wbrou@uab.edu.
† Department of Chemistry.
‡ Department of Vision Sciences.
§ Department of Optometry.
| Center for Biophysical Sciences and Engineering.
a Abbreviations: ADH, alcohol dehydrogenase; ATP, adenosine tri-
phosphate; BOG, â-(n-octyl)-D-glucopyranoside; CMC, critical micellar
concentration; CFU, colony forming unit; HEPPS, 3-[4-(2-hydroxyethyl)-
1-piperazinyl]propanesulfonic acid; MIC, minimum inhibitory concentration;
NaAD, nicotinic acid adenine dinucleotide; NAD, â-nicotinamide adenine
dinucleotide; NADH, â-nicotinamide adenine dinucleotide, reduced form.
10.1021/jm061349l CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/10/2007

Antibacterial Nicotinamide Adenine Dinucleotide
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2613
(b) Guanidines. The structures of the above compounds
contain a permanent positive charge, which is unfavorable for
membrane permeability and oral bioavailability of potential
drugs. Thus, we next proposed compounds 13a-d (Figure 3),
which are analogs of 11a-d but contain a highly basic
guanidinium group in place of the trimethylammonium group.
Note that the guanidinium group does not contain a permanent
positive charge.
The syntheses of guanidine analogs 13a-d are shown in
Scheme 3. Thus, amines 10a-d were treated with 1,3-bis(Boc)-
S-methylisothiourea in the presence of HgCl2 to give protected
guanidines 12a-d. Removal of the Boc groups using TFA
followed by treatment with 1 N HCl afforded the hydrochloride
salts of guanidines 13a-d.
Compounds 13a-d were evaluated as inhibitors of B. subtilis
NAD synthetase and as antibacterial agents. As shown in Table
1, IC50 and MIC values again remained similar to the earlier
tethered dimers discussed above. However, it is important to
note that the N,N-dimethyl (tertiary amine) analogs of 1, 2a-
2c, and 11a-11d are ineffective either as inhibitors of NAD
synthetase (up to 200 µM) or as antibacterial agents (MIC >
50 µg/mL; data not shown). Thus, either a permanent positive
charge, or a positive charge generated from a strongly basic
functionality, appears to be a structural requirement for activity
in this series.
Studies on Nonspecific Detergent Effects and Promiscuous
Inhibition. Compounds 1, 2a-2c, 11a-11d, and 13a-13d all
display remarkably similar activities as both NAD synthetase
inhibitors and antibacterial agents. While indeed these com-
pounds are structurally very similar, this observation raised
concerns regarding possible nonspecific actions that may cause
the observed biological activities. Specifically, we considered
the possibility that tethered dimers containing positively charged
end groups may act through a (a) nonspecific detergent effect
and/or (b) may show promiscuous behavior. Thus, we pursued
studies to better understand the mechanism of enzyme inhibition
by these compounds.
(a) Detergent Effects. Given the structural resemblance of
our best inhibitors to detergents, it was of particular interest to
determine if structurally similar (thus possessing similar deter-
gent properties) compounds would reveal significant differences
in enzyme activity. As we reported earlier,14 different positional
isomers, assumed to have nearly identical detergent properties,
sometimes exhibited very different enzyme inhibition activities.
This observation suggested that specific interactions give rise
to enzyme inhibition, as opposed to nonspecific detergent or
surfactant properties, although detergent properties at that time
were not directly measured. To further support this conclusion,
we have measured the CMC (critical micelle concentration)
values of three of the most active tethered dimer inhibitors (1,
2a, and 11a), which all have IC50 values in the low micromolar
range. CMC values were determined by two different experi-
mental methods, one using solubilization of a colored dye15 and
the second using fluorescence16 to detect micelle formation. The
measured CMC values by these two methods, compared to those
for known detergents, are shown in Table 2. As illustrated, while
these compounds are obviously good detergents, the CMC
values are at least 10-fold greater than the IC50 values. These
results suggest that detergent properties of these compounds
are not primarily responsible for their enzyme inhibition
activity.
Figure 1.
The protecting groups were removed using TFA to give the
free amines 6a-c, which were reacted with iodomethane to
provide the quaternary ammonium salts 2a-c.
The IC50 values of 2a-c for the inhibition of purified B.
subtilis NAD synthetase are given in Table 1. Compounds 2a-c
are all effective enzyme inhibitors as compared to compound
1. Compounds 2a and 2b were further tested against different
strains of Gram-positive and Gram-negative bacteria, and their
MIC (minimum inhibitory concentration) values were deter-
mined. For comparison purposes, identical MIC determinations
were performed with rifampin, methicillin, ampicillin, and
ciprofloxacin. The results are given in Table 1.
As shown, compounds 2a and 2b are potent inhibitors of the
growth of Gram-positive bacteria but are inactive against Gram-
negative bacteria (e.g., Pseudomonas). They are similar in
effectiveness to commercial antibiotics. Of particular interest
is the observation that methicillin-resistant strains of S. aureus
also remain susceptible.
All tethered dimer inhibitors that we have described so far
contain an ester linkage. We have observed (unpublished results)
that these esters are susceptible to plasma esterases. To
circumvent this problem, we proposed a common strategy of
changing the ester to a more stable amide or ether functionality.
Thus, the syntheses of amide- or ether-linked compounds 11a-d
(Figure 2) was next pursued. The procedure is described in
Scheme 2. As shown, for the amide linkers, 4-benzyloxyphenol
(3) was alkylated with N-(8-bromooctyl) phthalimide and NaH
to give phthalimide 7. The phthalimide group was removed by
treatment with hydrazine to provide amine 8 as its hydrochloride
salt. Amine 8 was converted to the intermediate amides 9a,b
by treatment with (L)-N-(tert-butoxycarbonyl)phenylalanine or
(L)-N-(tert-butoxycarbonyl)phenylglycine, respectively, in CH2-
Cl2 in the presence of EDAC and DMAP.
For the ether linkers, intermediate alcohol 4 (Scheme 1) was
converted to the triflate using triflic anhydride, and the product,
in one pot, was converted to ethers 9c,d by treatment with (L)-
N-(tert-butoxycarbonyl)phenylalaninol or (L)-N-(tert-butoxy-
carbonyl)phenylglycinol in the presence of NaH.
The final amide and ether targets 11a-d were prepared by
removal of the Boc group from 9a-d using TFA to afford
amines 10a-d. The reaction of amines 10a-d with io-
domethane gave the final quaternary ammonium products
11a-d.
Compounds 11a-d were evaluated as inhibitors of B. subtilis
NAD synthetase, and the IC50 values are given in the Table 1.
As shown, compounds 11a-d all effectively inhibit the enzyme.
Compounds 11a and 11b are the amide- and ether-linked
analogs of parent ester 2a, and this structural change had little
effect on activity. Also, shortening of the amino acid end to
phenylglycine (amide 11c and ether 11d) similarly had little
effect, revealing that the amide and ether linkages provide
tethered dimers with comparable activity to the parent esters.
Further, as found for 2a, compounds 11a-d were effective
inhibitors of the growth of Gram-positive bacteria but were
inactive against the Gram-negative Pseudomonas aeruginosa.
(b) Promiscuous Inhibition. “Promiscuous inhibitors” have
recently been described as compounds that nonspecifically
inhibit a variety of enzymes through the formation of aggregates

2614 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Velu et al.
Scheme 1
Table 1. Inhibition of B. Subtilis NAD Synthetase (IC50, µM), and Antibacterial Activities (MIC, µM) for Tethered Dimers.
MIC (µM)
Staphylococcus
Staphylococcus
aureus
aureus
Pseudomonas
aeruginosa
IC50
Bacillus subtilis
(MRSAa)
cmpd
(µM)
ATCC# 14289
ATCC# 29213
ATCC# 33591
ATCC# 27853
1
2a
2b
2c
41 ( 8.5
22 ( 2.4
53 ( 9.5
19 ( 2.1
40 ( 4.5
21 ( 1.2
45 ( 0.8
38 ( 6.1
12 ( 1.8
11 ( 2.2
19 ( 0.6
11 ( 1.1
2
0.78
1.56
9.4
6.2
0.78
>50
0.78
>50
b
b
3.13
b
b
b
b
11a
11b
11c
11d
13a
13b
13c
13d
doxycycline
ampicillin
ciprofloxacin
rifampin
0.39
0.39
1.56
0.78
3.13
3.13
1.56
3.13
1.56
0.2
0.78
0.78
0.78
1.56
1.56
3.13
1.56
0.78
1.56
0.78
>30
>50
>50
>50
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
1.56
b
b
b
0.5
1.5
0.5
b
b
methicillin
1
32
a MRSA ) methicillin-resistant Staphylococcus aureus. b Data not available.
Figure 2.
Figure 3.
that are hypothesized to bind and inactivate enzyme.17-19 The
inhibition of promiscuous inhibitors can be greatly diminished
by the addition of a detergent such as Triton X-100. We
proposed to evaluate the promiscuous behavior of the tethered
dimers 2a and 11a by observing the effect of added detergent
on the inhibition of NAD synthetase and on a second enzyme,
alcohol dehydrogenase (ADH; used as a reporter enzyme in the
NADS assay). We selected Triton X-100 as the added detergent,
because it has been shown to diminish the inhibitory activities
of some known promiscuous inhibitors at concentrations as low
as 0.001%, and most promiscuous inhibitors are reported to lose
inhibition in the presence of 0.01% Triton X-100.18 The latter

Antibacterial Nicotinamide Adenine Dinucleotide
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2615
Table 2. CMC Values of Selected Inhibitors
likely others) appear to exhibit classical behavior. Further, while
the present and earlier studies are consistent with NAD
synthetase inhibition as the target for antibacterial actions, other
unexplored mechanisms of action remain as possibilities.
CMC (mM)
cmpd
2a
ink
0.35
fluorescence
0.14
0.65
11a
0.45
Experimental Section
CTAB
BOG
SDS
0.8 (0.9a)
21 (19a)
Enzyme Assays. High-Throughput Assay for NAD Synthetase
Inhibition. NAD synthetase from Bacillus subtilis was overex-
pressed and purified as previously described.12 We also previously
reported12 the detailed procedure for high-throughput inhibition
assays. Briefly, this coupled assay monitors the production of NAD
in the enzyme reaction through its conversion to NADH in the
presence of ethanol and ADH from bakers’ yeast (Sigma). The assay
system at pH 8.5 contained 58.5 mM HEPPS buffer, 18.5 mM NH4-
Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, 0.1 mM
NaAD, 0.3% BOG, 40 µg/mL ADH, 2.0 µg/mL NAD synthetase,
0.2 mM ATP, and inhibitor at several concentrations (with 2.5%
(v/v) final DMSO concentration). The enzyme inhibition assay was
carried out in 96-well microtiter plates with a total reaction volume
of 200 µL. Two detection methods were simultaneously em-
ployed: UV absorbance at 340 nm and fluorescence emission at
460 nm, both resulting from the NADH produced in the reaction.
OD was read on a SpectraMax Pro microplate reader (Molecular
Devices), and fluorescence was read on a PolarStar microplate
reader (BMG-LabTechnologies). After initiation by adding 25 µL
of ATP solution, the reaction was allowed to proceed for 10 min
and stopped by adding 50 µL of 6 M guanidine‚HCl. The IC50 value
was determined by plotting the percentage inhibition versus
compound concentration.
7.5 (7.8a)
4.3 (3.5b)
TTABr
a Literature value.15 b Literature value.16
Table 3. Effect of Added Triton X-100 on the Inhibition of NAD
Synthetase
IC50 (µM)
0% Triton
X-100
0.001% Triton
X-100
0.01% Triton
X-100
cmpd
2a
11a
clotrimazole
16.0 ( 0.7
27.0 ( 2.8
>1500
16.5 ( 0.7
27.8 ( 1.1
no inhibition
37.0 ( 0.7
104.5 ( 3.5
no inhibition
Table 4. Effect of Added Triton X-100 on the Inhibition of ADH.
IC50 (µM)
0% Triton
X-100
0.001% Triton
X-100
0.01% Triton
X-100
cmpd
2a
11a
clotrimazole
27.0 ( 2.8
>1500
96.5 ( 16.3
69.0 ( 15.6
>1500
no inhibition
265.5 ( 6.4
>1500
no inhibition
Inhibition Assay for NAD Synthetase with Added Triton
X-100. The fluorescence-based assays were carried out in opaque
96-well assay plates using a PolarStar Optima fluorescence plate
reader from BMG LabTech. The final reaction volume was 200
µL in each well and contained the same components as described
above for the high-throughput assay, except the 0.3% BOG was
omitted. Additionally, 0 to 0.01% Triton X-100 was added along
with inhibitors 2a, 11a, and clotrimazole at various concentrations.
The assay was initiated by adding 0.2 mM ATP. The production
of NADH was monitored by fluorescence at an emission wavelength
of 460 nm (excitation wavelength was 355 nm) as a function of
time. The linear part of the kinetic curve was used to calculate the
initial reaction rate, Vo. Percentage inhibition was calculated based
on the difference in Vo with and without the inhibitors. The IC50
value was determined by plotting the percentage inhibition versus
the inhibitor concentration. Each point on the IC50 curve was an
average of three measurements. The final IC50 values were averages
from two sets of experiments.
Inhibition Assay for ADH with Added Triton X-100. The
reaction mixture contained 60 mM HEPPS, pH 8.5, 0.5 mM NH4-
Cl, 20 mM KCl, 10 mM MgCl2, 60 mM ethanol, 2.5 µg/mL yeast
ADH, 2.5% (v/v) DMSO, 0 to 0.01% Triton X-100, and inhibitors
2a, 11a, or clotrimazole at various concentrations. The assay was
initiated by adding 0.75 mM â-NAD and stopped by the addition
of 1.2 M guanidine‚HCl after 2 min. The production of NADH
was measured by fluorescence at an emission wavelength of 460
nm (excitation wavelength is 355 nm). Endpoint values were
calculated by subtracting fluorescence of the mixture before adding
â-NAD from fluorescence after adding guanidine‚HCl. The percent
inhibition was calculated based on the difference in endpoint values
with inhibitor and without inhibitor. Each point on the IC50 curve
was an average of three measurements. The final IC50 values were
averages from two sets of experiments.
concentration has little effect on enzyme activity or inhibition
by conventional inhibitors. We also included a reported18
classical promiscuous inhibitor, clotrimazole, which has been
shown to be a strong aggregator.
As shown in Table 3, the two tethered dimer NAD synthetase
inhibitors behaved similarly in the inhibition assay as the %
Triton X-100 increased from 0 to 0.01%. The IC50 values did
not increase with added 0.001% Triton X-100 but increased
somewhat as the detergent concentration was increased to
0.01%. However, in all cases, the increase in IC50 was no more
than 4-fold, which is significantly less dramatic than typically
observed for strong aggregators. Interestingly, clotrimazole was
not an inhibitor of NAD synthetase, suggesting this enzyme may
be less sensitive to promiscuous inhibitors.
The results for inhibition of ADH are summarized in Table
4. As shown, tethered dimer 11a did not inhibit up to the highest
concentration tested. However, dimer 2a was a significant
inhibitor of ADH, suggesting promiscuous behavior on this
enzyme. This was further supported by the 10-fold increase in
IC50 in the presence of 0.01% Triton X-100. Further, the
promiscuous inhibitor clotrimazole was, unlike the result with
NADS, an effective inhibitor of ADH (IC50 ) 96 µM), and
this inhibition disappeared at the lowest detergent concentration
(0.001%), which is characteristic of promiscuous inhibitors.
Thus, 2a appears to exhibit moderate promiscuous behavior on
ADH, although not as strongly as that seen for clotrimazole’s
inhibition of ADH. It is important to note that, while 2a inhibits
ADH in this promiscuous inhibition experiment, much higher
concentrations of ADH and ethanol were used in the NAD
synthetase assay, and 2a did not inhibit ADH when evaluated
under these conditions.
Dimer 2a was also evaluated for inhibition against ADH in a
separate set of experiments that used the same conditions as the
NAD synthetase coupled assay. The reaction mixtures consisted
of the same buffering components as the promiscuous study, along
with 0.3% BOG, 40 µg/mL ADH, 1% ethanol, and three different
concentrations of â-NAD (0.75 mM, 0.1 mM, and 0.01 mM). The
kinetic curves of the reactions were monitored by fluorescence after
initiation of the reaction by adding â-NAD. The reaction reached
In conclusion, dimers 2a and 11a do not exhibit promiscuous
behavior with NAD synthetase. Further, these compounds inhibit
NAD synthetase at concentrations well below their CMC values.
The results emphasize that, while contributions to the inhibition
of NAD synthetase from nonspecific behavior are possible for
some tethered dimer inhibitors, these selected examples (and

2616 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Velu et al.
Scheme 2
Scheme 3
Brucker 300 MHz spectrometer using TMS as internal standard.
The values of chemical shifts (δ) are given in ppm and coupling
constants (J) are given in Hz. Elemental analyses were performed
by Atlantic Microlab, Norcross, Georgia, and the results indicated
by symbols for the elements were within (0.4% of theoretical
values. Reactions were monitored by TLC (Whatmann, silica gel,
UV254, 25 µm plates), and flash column chromatography utilized
“Baker” silica gel (40 µm) in the solvent systems indicated.
Anhydrous solvents used for reactions were purchased in Sure-
Seal bottles from Aldrich Chemical Co. Other reagents were
purchased from Aldrich, Lancaster, or Acros chemical companies
and used as received.
8-(4-Benzyloxyphenoxy)octan-1-ol (4). To a solution of 4-ben-
zyloxyphenol 3 (5.0 g, 25 mmol) in anhydrous DMF (40 mL), NaH
(0.83 g, 28 mmol) was added and the mixture was stirred at room
temperature for 30 min. A solution of 8-bromo-1-octanol (4.7 mL,
27 mmol) in anhydrous DMF (20 mL) was added and the mixture
was stirred at room temperature for 3 h. The reaction mixture was
quenched with saturated NH4Cl (50 mL) and extracted with EtOAc
(3 × 80 mL). The combined EtOAc extracts were washed with
water (3 × 80 mL) and brine (1 × 80 mL) and dried over Na2SO4.
The drying agent was removed by filtration and the filtrate was
evaporated under vacuum to afford the crude product, which
crystallized upon the addition of hexanes. This was filtered,
washed with hexanes, and dried to obtain pure 4 (6.6 g, 80%): mp
at least 60% completion before the first point of the curve could
be read (within 15 s) and reached a plateau within 1 min, regardless
of the â-NAD concentration. Dimer 2a did not show any inhibition
in this study.
Antibacterial Assays. The detailed procedure was previously
reported.12 As before, MICs were determined by MicroBioTest, Inc.,
of Sterling, VA. Briefly, bacteria were subcultured from stock
cultures onto the appropriate agar and incubated overnight at 37 (
2 °C in ambient air. The overnight cultures were inoculated into 3
mL of the appropriate broth, and each suspension was adjusted to
contain approximately 5 × 108 CFU/mL. Starting at 50 µg/mL
inhibitor, doubling dilutions were created for a total of 10 tubes
(in duplicate), and each tube was then inoculated with the challenge
microorganism broth (0.01 mL). The tubes were incubated at 37
( 2 °C for 20 h. The MIC was defined as the lowest concentration
of test substance that completely inhibited visible growth of the
microorganism. Controls were included for viability, sterility, and
reproducible MIC values for known antibiotics.
1
94-95 °C; H NMR (CDCl3) δ 1.28-1.51 (m, 8H), 1.51-1.63
(m, 3H), 1.69-1.81 (m, 2H), 3.62 (t, 2H, J ) 6.58 Hz), 3.88 (t,
2H, J ) 6.52 Hz), 5.00 (s, 2H), 6.82 (d, 2H, J ) 9.09 Hz), 6.89 (d,
2H, J ) 9.21 Hz), 7.26-7.45 (m, 5H); 13C NMR (CDCl3) δ 25.6,
25.9, 29.3, 32.6, 62.9, 68.5, 70.6, 115.3, 115.7, 127.4, 127.8, 128.4,
137.2, 152.7, 153.4; IR (KBr) 3303 cm-1; MS (ES) m/z 329 (M +
H); Anal. (C21H28O3) C, H.
8-(4-Benzyloxyphenoxy)-1-octyl 2-(N-tert-Butoxycarbonyl-
amino)-3-phenylpropionate (5a). To a solution of alcohol 4 (0.328
g, 1.00 mmol) in anhydrous CH2Cl2 (20 mL), N-Boc-phenylalanine
(0.318 g, 1.20 mmol), EDAC (0.229 g, 1.12 mmol), and DMAP
(0.012 g, 0.10 mmol) were added and the mixture was stirred at
room temperature for 3 h. This was diluted with 10 mL of CH2Cl2
Synthetic Chemistry. General. Melting points were determined
using an Electrothermal 9100 apparatus and are uncorrected. IR
spectra were recorded with Brucker Vector-22 and Bomen MB-
104 instruments. All 1H and 13C NMR spectra were recorded on a

Antibacterial Nicotinamide Adenine Dinucleotide
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2617
and washed with 1 M NaHCO3 (3 × 10 mL), water (2 × 10 mL),
and brine (1 × 10 mL) and dried over Na2SO4. The drying agent
was removed by filtration and the filtrate was evaporated under
vacuum to afford the crude product, which was purified by flash
column chromatography on silica gel (2 × 10 cm; CHCl3 eluent)
to afford pure 5a (0.495 g, 86.1%): 1H NMR (CDCl3) δ 1.25-
1.44 (m, 8H), 1.41 (s, 9H), 1.51-1.65 (m, 2H), 1.69-1.78 (m,
2H), 2.98-3.10 (m, 2H), 3.89 (t, 2H, J ) 6.5 Hz), 4.10 (t, 2H, J
) 6.5 Hz), 4.51-4.62 (m, 1H), 4.97 (bs, 1H, NH), 5.01 (s, 2H),
6.82 (d, 2H, J ) 9.2 Hz), 6.90 (d, 2H, J ) 9.2 Hz), 7.10-7.45 (m,
10H); 13C NMR (CDCl3) δ 25.7, 25.9, 28.2, 28.4, 29.1, 29.2, 29.3,
38.4, 54.4, 65.4, 68.4, 70.6, 79.8, 115.3, 115.7, 126.9, 127.4, 127.8,
128.4, 128.5, 129.2, 136.0, 137.2, 152.8, 153.4, 155.0, 171.9; MS
(ES) m/z 576 (M + H); Anal. (C35H45NO6) C, H, N.
8-(4-Benzyloxyphenoxy)-1-octyl 2-(N-tert-Butoxycarbonyl-
amino)-3-(4-tert-butoxyphenyl)propionate (5b). Compound 5b
(0.66 g, 100%) was prepared using a procedure similar to that for
5a: 1H NMR (CDCl3) δ 1.25-1.45 (m, 8H), 1.31 (s, 9H), 1.41 (s,
9H), 1.46-1.63 (m, 2H), 1.70-1.79 (m, 2H), 2.94-3.08 (m, 2H),
3.88 (t, 2H, J ) 6.5 Hz), 4.01-4.11 (m, 2H), 4.50-4.58 (m, 1H),
4.97 (br s, 1H, NH), 4.99 (s, 2H), 6.81 (d, 2H, J ) 9.2 Hz), 6.89
(d, 2H, J ) 9.2 Hz), 6.90 (d, 2H, J ) 8.4 Hz), 7.02 (d, 2H, J ) 8.4
Hz), 7.27-7.44 (m, 5H); 13C NMR (CDCl3) δ 25.6, 25.9, 28.2,
28.3, 28.7, 29.0, 29.1, 29.2, 37.7, 54.4, 65.3, 68.3, 70.5, 78.2, 79.7,
115.2, 115.6, 124.0, 127.3, 127.7, 128.4, 129.6, 130.8, 137.2, 152.7,
153.3, 154.2, 155.0, 171.9; MS (ES) m/z 648 (M + H).
8-(4-Benzyloxyphenoxy)-1-octyl 2-(N-tert-Butoxycarbonyl-
amino)-3-(1H-indol-3-yl)propionate (5c). Compound 5c (0.52 g,
84.1%) was prepared using a procedure similar to that for 5a: 1H
NMR (CDCl3) δ 1.18-1.48 (m, 10H), 1.42 (s, 9H), 1.48-1.60 (s,
2H), 1.74 (p, 2H, J ) 6.5 Hz), 3.27 (d, 2H, J ) 5.43 Hz), 3.89 (t,
2H, J ) 6.5 Hz), 3.99-4.06 (m, 2H), 4.59-4.66 (m, 1H), 5.00 (s,
2H), 5.09 (br d, 1H, NH, J ) 7.7 Hz), 6.82 (d, 2H, J ) 9.1 Hz),
6.90 (d, 2H, J ) 9.1 Hz), 6.95 (s, 1H), 7.07-7.56 (m, 9H), 8.23
(br s, 1H, NH); 13C NMR (CDCl3) δ 25.6, 25.9, 28.1, 28.3, 28.4,
29.0, 29.1, 29.3, 54.2, 65.4, 68.5, 70.6, 79.7, 110.1, 111.1, 115.3,
115.7, 118.7, 119.4, 122.1, 122.7, 127.4, 127.6, 127.8, 128.5, 136.1,
137.2, 152.8, 153.4, 155.2, 172.4; MS (ES) m/z 615 (M + H).
8-(4-Benzyloxyphenoxy)-1-octyl 2-Amino-3-phenylpropionate
(6a). To a solution of the ester 5a (0.38 g, 0.66 mmol) in CH2Cl2
(2 mL), a mixture of TFA (2 mL) and CH2Cl2 (2 mL) was added
dropwise and the solution was stirred at room temperature for 30
min. The solvent and TFA were completely removed under vacuum
and the residue was dissolved in CH2Cl2 (20 mL). This solution
was washed with 1 M Na2CO3 (3 × 10 mL), water (2 × 10 mL),
and brine (1 × 10 mL) and dried over Na2SO4. The drying agent
was removed by filtration and the filtrate was evaporated in vacuo
to afford the crude product, which was purified by flash column
chromatography on silica gel (2 × 10 cm) using MeOH/CHCl3
eluent (1:19) to afford pure 6a (0.26 g, 83%): 1H NMR (CDCl3)
δ 1.25-1.39 (m, 6H), 1.39-1.51 (m, 4H), 1.51-1.64 (m, 2H, NH2),
1.73 (p, 2H, J ) 6.5 Hz), 2.85 (dd, 1H, J1 ) 13.5 Hz, J2 ) 7.8
Hz), 3.05 (dd, 1H, J1 ) 13.5 Hz, J2 ) 5.5 Hz), 3.70 (dd, 1H, J1 )
7.8 Hz, J2 ) 5.5 Hz); 3.86 (t, 2H, J ) 6.5 Hz), 4.07 (t, 2H, J ) 6.5
Hz); 4.98 (s, 2H), 6.80 (d, 2H, J ) 9.2 Hz), 6.88 (d, 2H, J ) 9.2
Hz), 7.16-7.41 (m, 10H); 13C NMR (CDCl3) δ 25.6, 25.8, 28.3,
28.9, 29.0, 29.1, 41.0, 55.6, 64.8, 68.2, 70.4, 115.1, 115.5, 126.6,
127.2, 127.6, 128.3(2C), 129.1(2C), 137.1, 152.6, 153.2, 174.9; MS
(ES) m/z 476 (M + H); Anal. (C30H37NO4) C, H, N.
130.2, 137.1, 152.7, 153.3, 155.4, 174.8; MS (ES) m/z 492 (M +
H); Anal. (C30H37NO5) C, H, N.
8-(4-Benzyloxyphenoxy)-1-octyl 2-Amino-3-(1H-indol-3-yl)-
propionate (6c). Compound 6c (0.25 g, 74.6%) was prepared using
a procedure similar to that for 6a: 1H NMR (CDCl3) δ 1.19-1.44
(m, 10H), 1.44-1.62 (m 2H), 1.63-1.80 (m, 2H), 1.96 (br s, 2H,
NH2), 3.01 (dd, 1H, J1 ) 14.2 Hz, J2 ) 7.6 Hz), 3.25 (dd, 1H, J1
) 14.2 Hz, J2 ) 4.6 Hz), 3.75-3.83 (m, 1H), 3.85 (t, 2H, J ) 5.3
Hz), 4.06 (t, 2H, J ) 6.2 Hz), 4.94 (s, 2H), 8.79 (d, 2H, J ) 9.1
Hz), 6.87 (d, 2H, J ) 9.1 Hz), 6.89 (s, 1H), 7.05-7.39 (m, 8H),
7.58 (d, 1H, J ) 7.6 Hz), 8.71 (br s, 1H, NH); 13C NMR (CDCl3)
δ 25.5, 25.7, 28.2, 28.9, 29.0, 29.1, 30.7, 54.8, 64.9, 68.2, 70.4,
110.5, 111.1, 115.1, 115.5, 118.4, 119.1, 121.7, 122.9, 127.24,
127.29, 127.6, 128.3, 136.1, 137.1, 152.6, 153.2, 175.2; MS (ES)
m/z 515 (M + H); Anal. (C32H38N2O4) C, H, N.
8-(4-Benzyloxyphenoxy)-1-octyl 2-(N,N,N-Trimethylammo-
nium)-3-phenylpropionate, Iodide (2a). To a solution of the amine
6a (0.150 g, 0.315 mmol) in DME (5 mL) was added K2CO3 (0.260
g, 1.88 mmol) followed by iodomethane (0.200 mL, 3.21 mmol)
and the mixture was stirred at room temperature for 12 h.
Precipitation of the product quaternary ammonium salt occurred.
The precipitate was dissolved by the addition of CH2Cl2 (20 mL),
and K2CO3 was removed by filtration through celite-521. The filtrate
was concentrated and the product was precipitated by the addition
of hexanes. The precipitated product was filtered, washed on the
filter with cold EtOAc, and dried to obtain pure 2a (0.114 g,
58.6%): 1H NMR (CDCl3) δ 0.91-1.02 (m, 2H), 1.11-1.45 (m,
9H), 1.68-1.79 (m, 2H), 3.12 (t, 1H, J ) 12.1 Hz), 3.62-3.65
(m, 1H), 3.69 (s, 9H), 3.89 (t, 2H, J ) 6.6 Hz), 3.91 (t, 2H, J )
6.8 Hz), 4.71 (dd, 1H, J1 ) 12.1 Hz, J2 ) 4.2 Hz), 5.00 (s, 2H),
6.82 (d, 2H, J ) 9.2 Hz), 6.90 (d, 2H, J ) 9.2 Hz), 7.24-7.44 (m,
10H); 13C NMR (CDCl3) δ 25.2, 25.8, 27.7, 28.8, 28.9, 29.2, 33.3,
53.1, 66.9, 68.4, 70.6, 75.5, 115.2, 115.7, 127.4, 127.7, 128.0, 128.4,
129.0, 129.6, 131.8, 137.2, 152.7, 153.3, 166.6; MS (ES) m/z 518
(M+); Anal. (C33H44INO4) C, H, N.
8-(4-Benzyloxyphenoxy)-1-octyl 2-(N,N,N-Trimethylammo-
nium)-3-(4-hydroxyphenyl)propionate, Iodide (2b). Compound
2b (0.105 g, 60.0%) was prepared using a procedure similar to
that for 2a: 1H NMR (CDCl3) δ 1.01-1.46 (m, 10H), 1.71 (p, 2H,
J ) 6.8 Hz), 2.97 (t, 1H, J ) 12.1 Hz), 3.45 (s, 9H), 3.46-3.55
(m, 1H), 3.85-4.00 (m, 4H), 4.55-4.59 (m, 1H), 4.97 (s, 2H),
6.81 (d, 2H, J ) 9.2 Hz), 6.88 (d, 2H, J ) 9.2 Hz), 6.95 (d, 2H,
J ) 8.2 Hz), 7.07 (d, 2H, J ) 8.2 Hz), 7.26-7.41 9 m, 5H), 7.50
(s, 1H, OH); 13C NMR (CDCl3) δ 25.2, 25.8, 27.8, 28.8, 28.9, 29.1,
32.5, 53.1, 66.9, 68.5, 70.5, 75.5, 115.3, 115.6, 116.1, 122.9, 127.3,
127.7, 128.4, 130.7, 137.1, 152.7, 153.1, 156.2, 166.5; MS (ES)
m/z 534 (M+); Anal. (C33H44INO5) C, H, N.
8-(4-Benzyloxyphenoxy)-1-octyl 2-(N,N,N-Trimethylammo-
nium)-3-(1H-indol-3-yl)propionate, Iodide (2c). Compound 2c
(0.135 g, 67.6%) was prepared using a procedure similar to that
for 2a: 1H NMR (CDCl3) δ 0.75-0.90 (m, 2H), 0.99-1.19 (m,
6H), 1.31-1.41 (m, 2H), 1.70 (p, 2H, J ) 7.02 Hz), 3.23-3.33
(m, 1H), 3.41 (s, 9H), 3.55-3.65 (m, 1H), 3.72-3.81 (m, 2H),
3.88 (t, 2H, J ) 6.5 Hz), 4.46-4.53 (m, 1H), 4.99 9s, 2H), 6.82
(d, 2H, J ) 9.1 Hz), 8.90 (d, 2H, J ) 9.1 Hz), 7.04-7.16 (m, 2H),
7.27-7.48 (m, 7H), 9.36 (s, 1H); 13C NMR (CDCl3) δ 23.4, 25.0,
25.8, 27.5, 28.7, 28.9, 29.2, 52.9, 67.0, 68.5, 70.5, 75.1, 105.2,
111.8, 115.3, 115.7, 118.0, 119.7, 122.1, 124.8, 126.4, 127.4, 127.8,
128.4, 135.8, 137.1, 152.7, 153.3, 167.1; MS (ES) m/z 557 (M+);
Anal. (C35H45IN2O4‚0.25H2O) C, H, N.
8-(4-Benzyloxyphenoxy)-1-octyl 2-Amino-3-(4-hydroxyphen-
yl)propionate (6b). Compound 6b (0.31 g, 100%) was prepared
using a procedure similar to that for 6a: 1H NMR (CDCl3) δ 1.27-
1.50 (m, 8H), 1.58-1.69 (m, 2H), 1.74 (p, 2H, J ) 6.5 Hz), 2.81
(dd, 1H, J1 ) 13.7 Hz, J2 ) 7.6 Hz), 3.02 (dd, 1H, J1 ) 13.7 Hz,
J2 ) 5.2 Hz), 3.61 (br s, 2H, NH2), 3.70 (dd, 1H, J1 ) 7.6 Hz, J2
) 2 Hz), 3.89 (t, 2H, J ) 6.5 Hz), 4.10 (t, 2H, J ) 6.5 Hz), 4.99
(s, 2H), 6.65 (d, 2H, J ) 8.5 Hz), 6.82 (d, 2H, J ) 9.2 Hz), 6.89
(d, 2H, J ) 9.2 Hz), 6.98 (d, 2H, J ) 8.5 Hz), 7.25-743 (m, 5H);
13C NMR (CDCl3) δ 25.7, 25.9, 28.4, 29.0, 29.1, 29.2, 39.7, 55.4,
65.3, 68.5, 70.6, 115.3, 115.6, 115.7, 127.4, 127.6, 127.8, 128.4,
N-[8-(4-Benzyloxyphenoxy)-1-octyl] Phthalimide (7). To a
solution of phenol 3 (8.00 g, 40.0 mmol) in anhydrous DMF (100
mL) under nitrogen, NaH (1.92 g, 60% in mineral oil, 48.0 mmol)
was added and the mixture was stirred at room temperature for 30
min. A solution of N-(8-bromooctyl)phthalimide (14.9 g, 44.0
mmol) in DMF (30 mL) was added dropwise over 5 min and the
reaction mixture was stirred at room temperature for 3 h. The
reaction was quenched with saturated NH4Cl (150 mL) and
extracted with EtOAc (3 × 150 mL). The organic layers were
combined, washed with 1 N NaOH (2 × 150 mL), water (3 × 150
mL), and brine (1 × 150 mL), and dried over Na2SO4. The drying

2618 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Velu et al.
agent was removed by filtration and the filtrate was evaporated in
vacuo to afford the crude product, which crystallized upon the
addition of hexanes. This was filtered, dried, and recrystallized from
methanol to afford the pure 7 (14.6 g, 79.8%): mp 72-73 °C; 1H
NMR (CDCl3) δ 1.25-1.50 (m, 8H), 1.65-1.79 (m, 4H), 3.67 (t,
2H, J ) 7.4 Hz), 3.88 (t, 2H, J ) 6.5 Hz), 5.00 (s, 2H), 6.82 (d,
2H, J ) 9.1 Hz), 6.89 (d, 2H, J ) 9.1 Hz), 7.25-7.45 (m, 5H),
7.67-7.72 (m, 2H), 7.81-7.86 (m, 2H); 13C NMR (CDCl3) δ 25.9,
26.7, 28.5, 29.0, 29.1, 29.2, 37.9, 68.4, 70.6, 115.3, 115.7, 123.1,
127.4, 127.8, 128.4, 132.1, 133.8, 137.3, 152.7, 153.4, 168.4; IR
(neat) 2929 cm-1; MS (ES) m/z 458 (M + H); Anal. (C29H31NO4)
C, H, N.
g, 3.04 mmol) in CH2Cl2 (40 mL) at 0 °C was added 2,6-lutidine
(0.460 mL, 3.95 mmol) followed by triflic anhydride (0.620 mL,
3.68 mmol). The addition was carried out slowly while maintaining
the temperature of the reaction at 0 °C. After stirring at 0 °C for
15 min, the reaction mixture was washed with water (2 × 20 mL)
and brine (1 × 20 mL) and dried over Na2SO4. After removal of
the drying agent, solvent was completely removed to obtain the
product triflate. This was dried at high vacuum for 15 min and
used without further purification for the next step. The crude triflate
was then dissolved in CH2Cl2 (10 mL) and added over 10 min to
a solution of N-Boc phenylalaninol (1.53 g, 6.09 mmol) and NaH
(0.305 g, 60% in mineral oil, 7.62 mmol) in CH2Cl2 (30 mL) at
0 °C. The reaction bubbled vigorously. It was stirred for 5 min
and then 18-crown-6 (0.081 g, 0.31 mmol) was added. The reaction
mixture was stirred at room temperature for another 30 min. The
reaction was then carefully quenched with water (20 mL) and the
organic layer was separated. The organic layer was washed with
water (2 × 20 mL) and brine (1 × 20 mL) and dried over Na2SO4.
The drying agent was filtered and the solvent was completely
removed under vacuum to obtain the crude product. It was purified
by silica flash column chromatography (4 × 20 cm) using EtOAc/
hexanes (1:9) as eluent to afford pure 9c (4.50 g, 67.5%): 1H NMR
(CDCl3) δ 1.28-1.36 (m, 6H), 1.41 (s, 9H), 1.43-1.51 (m, 2H),
1.51-1.64 (m, 2H), 1.69-1.81 (m, 2H), 2.75-2.94 (m, 2H), 3.23-
3.32 (m, 2H), 3.32-3.45 (m, 2H), 3.88 (t, 2H, J ) 6.5 Hz), 3.88-
3.96 (m, 1H), 4.88 (br d, 1H, J ) 8.04 Hz, NH), 4.98 (s, 2H), 6.81
(d, 2H, J ) 9.2 Hz), 6.89 (d, 2H, J ) 9.2 Hz), 7.15-7.43 (m,
10H); 13C NMR (CDCl3) δ 25.9, 26.1, 28.3 (2C), 29.2, 29.3, 29.5,
37.7, 51.5, 68.4, 70.2, 70.5, 71.1, 79.1, 115.2, 115.6, 126.1, 127.3,
127.7, 128.2, 128.4, 129.3, 137.2, 138.2, 152.7, 153.3, 155.2; IR
(neat) 3373, 1685 cm-1; MS (ES) 562 (M + H); Anal. (C35H47-
NO5) C, H, N.
1-(4-Benzyloxyphenoxy)-8[2-(N-tert-butoxycarbonylamino)-
2-phenyl-1-ethyloxy]octane (9d). Compound 9d (0.411 g, 75%)
was prepared using a procedure similar to that for 9c: 1H NMR
(CDCl3) δ 1.23-1.35 (m, 8H), 1.41 (s, 9H), 1.48-1.61 (m, 2H),
1.74 (p, 2H, J ) 6.6 Hz), 3.31-3.49 (m, 2H), 3.49-3.69 (m, 2H),
3.88 (t, 2H, J ) 6.6 Hz), 4.79 (br s, 1H), 5.01 (s, 2H), 5.26 (br s,
1H), 6.82 (d, 2H, J ) 9.2 Hz), 6.90 (d, 2H, J ) 9.2 Hz), 7.19-
7.45 (m, 10H); 13C NMR (CDCl3) δ 25.9 (2C), 28.3 (2C), 29.3
(2C), 29.4, 54.0, 68.4, 70.6, 71.2, 73.5, 79.4, 115.3, 115.7, 126.6,
127.1, 127.4, 127.8, 128.3, 128.5(2C), 137.2, 152.7, 153.4, 155.4;
IR (neat) 3382, 1687 cm-1; MS (ES) 548 (M + H); Anal. (C34H45-
NO5) C, H, N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-amino-3-phenylpropi-
onamide (10a). To a solution of amide 9a (0.400 g, 0.696 mmol)
in CH2Cl2 (2 mL) was added a mixture of TFA (2 mL) and CH2-
Cl2 (2 mL), dropwise, and the mixture was stirred at room
temperature for 30 min. The solvent and TFA were completely
removed under vacuum, and the residue was dissolved in CH2Cl2
(20 mL). The solution was washed with 1 M Na2CO3 (3 × 10 mL),
water (2 × 10 mL), and brine (1 × 10 mL) and dried over Na2-
SO4. The drying agent was removed by filtration and the filtrate
was evaporated in vacuo to afford the crude product, which was
purified by flash column chromatography on silica gel (2 × 10
cm) using MeOH/CHCl3 (1:19) eluent to afford pure 10a (0.310 g,
93.8%): 1H NMR (CDCl3) δ 1.23-1.61 (m, 14H including NH2),
1.74 (p, 2H, J ) 6.5 Hz), 2.69 (dd, 1H, J1 ) 13.6 Hz, J2 ) 9.3
Hz), 3.19-3.29 (m, 3H), 3.58 (dd, 1H, J1 ) 9.3 Hz, J2 ) 4.1 Hz),
3.89 (t, 2H, J ) 6.5 Hz), 5.00 (s, 2H), 6.82 (d, 2H, J ) 9.2 Hz),
6.89 (d, 2H, J ) 9.2 Hz), 7.18-7.44 (m, 10H); 13CNMR (CDCl3)
δ 25.9, 26.8, 29.1, 29.2, 29.3, 29.5, 39.0, 41.1, 56.4, 68.4, 70.6,
115.3, 115.7, 126.7, 127.4, 127.8, 128.4, 128.6, 129.2, 137.2, 137.7,
152.7, 153.4, 173.9; MS (ES) m/z 475 (M + H); Anal. (C30H38N2O3)
C, H, N.
8-(4-Benzyloxyphenoxy)-1-octylamine, Hydrochloride (8). To
a solution of phthalimide 7 (14.0 g, 30.6 mmol) in a mixture of
CH2Cl2 (175 mL) and MeOH (175 mL), hydrazine (5.88 g, 183.8
mmol) was added dropwise and the mixture was stirred at room
temperature under N2 for 12 h. A white precipitate of the byproduct
was filtered, the filtrate was evaporated in vacuo, and the residue
was taken up in a CHCl3/EtOAc (1:1) mixture (150 mL). More
precipitation of the byproduct occurred and it was again filtered.
The filtrate was evaporated under vacuum and the residue obtained
was dissolved in CHCl3 (200 mL). This was washed with water (2
× 100 mL) and brine (1 × 100 mL) and concentrated under vacuum
to a volume of 50 mL. HCl (1 N) was added dropwise with stirring
until the pH was ∼2. The white amine hydrochloride precipitated
and was filtered, washed on the filter with ice cold water, EtOAc,
and ether, and dried to obtain pure 8 (8.50 g, 76.4%): mp 185-
1
186 °C; H NMR (DMSO-d6) δ 1.22-1.44 (m, 8H), 1.48-1.61
(m, 2H), 1.66 (p, 2H, J ) 6.6 Hz), 2.67-2.81 (m, 2H), 2.69-2.78
(m, 2H), 3.86 (t, 2H, J ) 6.6 Hz), 5.02 (s, 2H), 6.83 (d, 2H, J )
9.1 Hz), 6.91 (d, 2H, J ) 9.1 Hz), 7.28-7.45 (m, 5H), 7.98 (br s,
3H, NH3); 13C NMR (DMSO-d6) δ 25.5, 25.8, 26.9, 28.5 (2C),
28.6, 28.7, 67.7, 69.6, 115.2, 115.6, 127.6, 127.7, 128.4, 137.3,
152.2, 152.8; IR (neat) 3440 cm-1; MS (ES) m/z 328 (M+); Anal.
(C21H30ClNO2) C, H, N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-(N-tert-butoxycarbonyl-
amino)-3-phenylpropionamide (9a). To a solution of amine
hydrochloride 8 (0.363 g, 1.00 mmol) in CH2Cl2 (10 mL), Et3N
(0.160 mL, 1.20 mmol) was added and the mixture was stirred at
room temperature for 5 min. Then N-(Boc)phenylalanine (0.397 g,
1.50 mmol), EDAC (0.286 g, 1.50 mmol), and DMAP (0.012 g,
0.10 mmol) were added and stirred at room temperature for 12 h.
The reaction mixture was diluted with CH2Cl2 (20 mL), washed
with 1 M NaHCO3 (3 × 10 mL), water (2 × 10 mL), and brine (1
× 10 mL), and dried over Na2SO4. The drying agent was removed
by filtration and the filtrate was evaporated under vacuum to afford
the crude product, which was purified by flash column chroma-
tography on silica gel (2 × 10 cm) using MeOH/CHCl3 (1:19)
eluent to afford pure 9a (0.410 g, 71.4%): 1H NMR (CDCl3) δ
1.11-1.49 (m, 10H), 1.40 (s, 9H), 1.67-1.77 (m, 2H), 2.95-3.18
(m, 4H), 3.88 (t, 2H, J ) 6.4 Hz), 4.19-4.32 (m, 1H), 5.00 (s,
2H), 5.12 (br s, 1H, NH), 5.74 (br s, 1H, NH), 6.81 (d, 2H, J )
9.2 Hz), 6.90 (d, 2H, J ) 9.2 Hz), 7.15-7.43 (m, 10H); 13C NMR
(CDCl3) δ 26.4, 27.1, 28.7, 29.5 (2C), 29.6, 29.7, 29.8, 39.1, 30.8,
68.9, 71.1, 80.5, 115.7, 116.2, 127.3, 127.8, 128.2, 128.9, 129.1,
129.7, 136.0, 137.7, 153.2, 153.8, 155.5, 171.2; MS (ES) m/z 575
(M + H); Anal. (C35H46N2O5) C, H, N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-(N′-tert-butoxycarbon-
ylamino)-2-phenylacetamide (9b). Compound 9b (0.41 g, 73%)
was prepared using a procedure similar to that for 9a: 1H NMR
(CDCl3) δ 1.07-1.29 (m, 6H), 1.29-1.47 (m, 4H), 1.40 (s, 9H),
1.70 (p, 2H, J ) 6.5 Hz), 3.02-3.26 (m, 2H), 3.85 (t, 2H, J ) 6.5
Hz), 4.98 (s, 2H), 5.14-5.39 (m, 1H), 5.87-6.14 (m, 1H), 6.37
(br s, 1H, NH), 6.80 (d, 2H, J ) 9.2 Hz), 6.89 (d, 2H, J ) 9.2 Hz),
7.24-7.43 (m, 10H); 13C NMR (CDCl3) δ 25.7, 26.4, 28.1, 28.9,
29.0, 29.2 (2C), 39.5, 58.1, 68.2, 70.4, 79.7, 115.2, 115.6, 126.9,
127.3, 127.7, 127.9, 128.3, 128.7, 137.1, 138.5, 152.6, 153.2, 155.1,
170.0; IR (neat) 3365, 3318, 1686, 1654 cm-1; MS (ES) 561 (M
+ H); Anal. (C34H44N2O5) C, H, N.
1-(4-Benzyloxyphenoxy)-8-(2-amino-3-phenyl-1-propyloxy)-
octane (10b). Compound 10b (0.31 g, 94.3%) was prepared using
a procedure similar to that for 10a: 1H NMR (CDCl3) δ 1.28-
1.50 (m, 8H), 1.50-1.64 (m, 2H), 1.73 (p, 2H, J ) 6.3 Hz), 2.47-
2.61 (m, 1H), 2.71-2.78 (m, 1H), 3.15-3.27 (m, 2H), 3.33-3.48
(m, 3H), 3.85 (t, 2H, J ) 6.3 Hz), 4.96 (s, 2H), 6.79 (d, 2H, J )
1-(4-Benzyloxyphenoxy)-8[2-(N-tert-butoxycarbonylamino)-
3-phenyl-1-propyloxy]octane (9c). To a solution of alcohol 4 (1.00

Antibacterial Nicotinamide Adenine Dinucleotide
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2619
9.1 Hz), 6.87 (d, 2H, J ) 9.1 Hz), 7.15-7.42 (m, 10H); 13CNMR
(CDCl3) δ 25.8, 25.9, 29.1(2C), 29.2, 29.5, 40.6, 52.2, 68.3, 70.4,
71.1, 75.2, 115.1, 115.5, 126.1, 127.2, 127.6, 128.2, 128.3, 129.1,
137.1, 138.8, 152.6, 153.3; MS (ES) m/z 462 (M + H); Anal.
(C30H39NO3) C, H, N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-amino-2-phenylacetam-
ide (10c). Compound 10c (0.41 g, 73.2%) was prepared using a
procedure similar to that for 10a: 1H NMR (CDCl3) δ 1.23-1.35
(m, 6H), 1.35-1.54 (m, 4H), 1.73 (p, 2H, J ) 6.5 Hz), 1.83 (br s,
2H, NH2), 3.24 (q, 2H, 7.1 Hz), 3.88 (t, 2H, J ) 6.5 Hz), 4.49 (s,
1H), 5.01 (s, 2H), 6.82 (d, 2H, J ) 9.2 Hz), 6.90 (d, 2H, J ) 9.2
Hz), 7.06 (br s, 1H, NH), 7.26-7.44 (m, 10H); 13CNMR (CDCl3)
δ 25.8, 26.7, 29.1, 29.2, 29.3, 29.5, 39.1, 59.8, 68.4, 70.6, 115.3,
115.7, 126.8, 127.4, 127.8, 127.9, 127.4, 128.7, 137.2, 141.1, 152.7,
153.3, 172.7; IR (neat) 3294, 1640 cm-1; MS (ES) 461 (M + H);
Anal. (C29H36N2O3) C, H, N.
1-(4-Benzyloxyphenoxy)-8-(2-amino-2-phenyl-1-ethyloxy)oc-
tane (10d). Compound 10d (0.21 g, 85.2%) was prepared using a
procedure similar to that for 10a: 1H NMR (CDCl3) δ 1.26-1.38
(m, 6H), 1.38-1.49 (m, 2H), 1.51-1.64 (m, 2H), 1.74 (p, 2H, J )
6.5 Hz), 1.92 (br s, 2H, NH2), 3.31-3.57 (m, 4H), 3.88 (t, 2H, J
) 6.5 Hz), 4.19 (dd, 1H, J1 ) 8.9 Hz, J2 ) 3.6 Hz), 4.99 (s, 2H),
6.82 (d, 2H, J ) 9.2 Hz), 6.89 (d, 2H, J ) 9.2 Hz), 7.21-7.44 (m,
10H); 13C NMR (CDCl3) δ 25.9, 26.1, 29.3 (2C), 29.4, 29.6, 55.4,
68.4, 70.6, 71.2, 115.3, 115.7, 126.7, 127.3, 127.4, 127.8, 128.3,
128.4, 137.2, 142.4, 152.7, 153.4; IR (neat) 3314-3162 cm-1; MS
(ES) 448 (M + H).
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-(N,N,N-trimethylam-
monium)-3-phenylpropionamide, Iodide (11a). To a solution of
the amine 10a (0.15 g, 0.32 mmol) in DME (5 mL) were added
K2CO3 (0.270 g, 1.95 mmol) and iodomethane (0.20 mL, 3.2 mmol),
and the mixture was stirred at room temperature for 12 h.
Precipitation of the product quaternary ammonium salt occurred,
and it was dissolved by the addition of CH2Cl2 (20 mL). K2CO3
was removed by filtration through celite-521. The filtrate was
concentrated and the product was precipitated upon the addition
of hexanes. This was filtered, washed on the filter with EtOAc,
and dried to obtain pure 11a (0.13 g, 66%): 1H NMR (CDCl3) δ
0.93-1.07 (m, 2H), 1.10-1.45 (m, 8H), 1.71 (p, 2H J ) 6.5 Hz),
2.85-2.95 (m, 2H), 3.06-3.34 (m, 3H), 3.48 (s, 9H), 3.87 (t, 2H,
J ) 6.5 Hz), 4.99 (s, 2H), 5.59 (dd, 1H, J1 ) 11.5 Hz, J2 ) 4.1
Hz), 6.82 (d, 2H, J ) 9.0 Hz), 6.89 (d, 2H, J ) 9.0 Hz), 7.22-
7.45 (m, 10H), 7.72 (t, 1H, J ) 5.8 Hz, NH); 13C NMR (CDCl3)
δ 25.8, 26.3, 28.2, 28.8, 28.9, 29.1, 32.8, 39.2, 52.6, 68.3, 70.5,
72.9, 115.2, 115.6, 127.3, 127.7, 128.4 (2C), 128.8, 129.6, 132.2,
137.1, 152.6, 153.3, 164.5; MS (ES) m/z 517 (M+); Anal. (C33H45-
IN2O3) C, H, N.
1-(4-Benzyloxyphenoxy)-8-(2-N,N,N-trimethylammonium-3-
phenyl-1-propyloxy)octane, Iodide (11b). Compound 11b (0.13
g, 60%) was prepared using a procedure similar to that for 11a:
1H NMR (CDCl3) δ 1.28-1.50 (m, 8H), 1.50-1.62 (m, 2H), 1.72-
1.81 (m, 2H), 3.05-3.42 (m, 5H), 3.57 (s, 9H), 3.84-3.88 (m,
1H), 3.90 (t, 2H, J ) 6.5 Hz), 4.26-4.35 (m, 1H), 5.01 (s, 2H),
6.82 (d, 2H, J ) 9.2 Hz), 6.90 (d, 2H, J ) 9.2 Hz), 7.21-7.43 (m,
10H); 13C NMR (CDCl3) δ 25.9, 26.0, 29.1, 29.20, 29.23, 29.3,
31.3, 53.4, 65.1, 68.3, 70.5, 71.7, 73.8, 115.2, 115.6, 127.3, 127.5,
127.7, 128.4, 129.0, 129.4, 134.7, 137.1, 152.7, 153.3; IR (neat)
2931, 2856 cm-1; MS (ES) 504 (M+); Anal. (C33H46INO3) C, H,
N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-(N,N,N-trimethylam-
monium)-2-phenylacetamide, Iodide (11c). Compound 11c (0.114
g, 58.1%) was prepared using a procedure similar to that for 11a:
1H NMR (CDCl3) δ 1.19-1.31 (m, 6H), 1.31-1.44 (m, 2H), 1.44-
1.60 (m, 2H), 1.63-1.76 (m, 2H), 3.17-3.32 (m, 2H), 3.38 (s,
9H), 3.85 (t, 2H, J ) 6.5 Hz), 4.99 (s, 2H), 6.70 (s, 1H), 6.80 (d,
2H, J ) 9.1 Hz), 6.88 (d, 2H, J ) 9.1 Hz), 7.26-7.56 (m, 8H),
7.85-7.94 (m, 3H, including NH); 13C NMR (CDCl3) δ 25.5, 26.4,
28.4, 28.6, 28.8, 28.9, 39.4, 51.8, 68.1, 70.3, 73.2, 115.0, 115.4,
126.6, 127.1, 127.5, 128.2, 129.0, 131.2, 131.8, 136.9, 152.4, 153.1,
164.6; IR (neat) 3223, 1679 cm-1; MS (ES) m/z 503 (M+); Anal.
(C32H43IN2O3‚0.5CHCl3) C, H, N.
1-(4-Benzyloxyphenoxy)-8-(2-N,N,N-trimethylammonium-2-
phenyl-1-ethyloxy)octane, Iodide (11d). Compound 11d (0.11 g,
78.1%) was prepared using a procedure similar to that for 11a: 1H
NMR (CDCl3) δ 1.31-1.51 (m, 8H), 1.57-1.69 (m, 2H), 1.69-
1.82 (m, 2H), 3.46 (s, 9H), 3.37-3.63 (m, 2H), 3.90 (t, 2H, J )
6.5 Hz), 4.06-4.14 (m, 1H), 4.20-4.25 (m, 1H), 5.01 (s, 2H), 5.44
(t, 1H, J ) 3.9 Hz), 6.82 (d, 2H, J ) 9.1 Hz), 6.91 (d, 2H, J ) 9.1
Hz), 7.27-7.53 (m, 8H), 7.73-7.79 (m, 2H); 13C NMR (CDCl3) δ
25.9, 26.1, 29.1 (2C), 29.2, 29.3, 53.1, 68.3, 69.5, 70.5, 72.1, 74.5,
115.3, 115.7, 127.3, 127.7, 128.4, 129.2, 130.8, 131.2 (2C), 137.1,
152.7, 153.3; IR (neat) 2934, 2858 cm-1; MS (ES) m/z 490 (M+);
Anal. (C32H44INO3‚0.5H2O) C, H, N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-(N′,N′′-bis-tert-butoxy-
carbonylguanidino)-3-phenylpropionamide (12a). To a solution
of amine 10a (6.00 g, 12.6 mmol) in anhydrous DMF (60 mL)
were added 1,3-bis(Boc)-2-methyl-2-thiopseudourea (3.67 g, 12.6
mmol) and Et3N (6.8 mL, 51 mmol), and the mixture was stirred
at room temperature for 5 min. HgCl2 (3.77 g, 13.9 mmol) was
added and formation of a white precipitate occurred almost instantly.
The mixture was stirred at room temperature for 30 min. It was
diluted with EtOAc (200 mL), filtered through celite-521 to remove
the precipitate, and the filter was washed with EtOAc (100 mL).
The filtrate was washed with 1 N NaOH (3 × 100 mL), water (2
× 100 mL), and brine (100 mL). Removal of solvent from the dried
(Na2SO4) extract gave the crude product, which was purified by
flash silica chromatography (7 × 20 cm) using EtOAc/hexanes (1:
4) as eluent to afford 12a as a colorless oil (8.21 g, 90.5%): 1H
NMR (CDCl3) δ 1.15-1.45 (m, 10H), 1.47 (s, 18H), 1.65-1.77
(m, 2H), 3.07-3.18 (m, 4H), 3.87 (t, 2H, J ) 6.5 Hz), 4.67 (q,
2H, J ) 7.2 Hz), 4.99 (s, 2H), 6.41 (t, 1H, NH, J ) 5.5 Hz), 6.81
(d, 2H, J ) 9.2 Hz), 6.89 (d, 2H, J ) 9.2 Hz), 7.19-7.44 (m,
10H), 8.81 (d, 1H, J ) 7.2 Hz), 11.31 (s, 1H, NH); 13C NMR
(CDCl3) δ 25.8, 26.5, 27.8, 28.1, 29.0 (2C), 29.1, 29.2, 37.5, 39.2,
55.7, 68.3, 70.5, 79.1, 83.3, 115.2, 115.6, 126.7, 127.3, 127.7, 128.3,
128.4, 129.3, 136.7, 137.1, 152.5, 152.6, 153.3, 155.6, 162.8, 169.9;
MS (ES) m/z 717 (M + H); Anal. (C41H56N4O7) C, H, N.
1-(4-Benzyloxyphenoxy)-8-(2-N,N′-bis-tert-butoxycarbon-
ylguanidino)-3-phenyl-1-propyloxy)octane (12b). Compound 12b
(0.218 g, 92.8%) was prepared using a procedure similar to that
for 12a: 1H NMR (CDCl3) δ 1.27-1.43 (m, 8H), 1.47 (s, 9H),
1.50 (s, 9H), 1.53-1.67 (m, 2H), 1.74 (p, 2H, J ) 6.5 Hz), 2.83-
3.02 (m, 2H), 3.25-3.35 (m, 2H), 3.39 (t, 2H, J ) 6.2 Hz), 3.87
(t, 2H, J ) 6.5 Hz), 4.41-4.54 (m, 1H), 4.98 (s, 2H), 6.81 (d, 2H,
J ) 9.2 Hz), 6.88 (d, 2H, J ) 9.2 Hz), 7.15-7.43 (m, 10H), 8.66
(d, 1H, J ) 8.3 Hz, NH), 11.5 (s, 1H, NH); 13C NMR (CDCl3) δ
25.9, 26.0, 27.9, 28.2, 29.2, 29.3, 29.4, 29.5, 37.2, 51.4, 68.3, 69.4,
70.4, 71.1, 78.7, 82.6, 115.2, 115.6, 126.2, 127.3, 127.7, 128.1,
128.3, 129.5, 137.1, 137.9, 152.6, 152.8, 153.3, 155.5, 163.6; MS
(ES) m/z 704 (M + H); Anal. (C41H57N3O7) C, H, N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-(N′,N′′-bis-tert-butoxy-
carbonylguanidino)-2-phenylacetamide (12c). Compound 12c
(0.113 g, 82.7%) was prepared using a procedure similar to that
for 12a: 1H NMR (CDCl3) δ 1.16-1.45 (m, 10H), 1.47 (s, 9H),
1.48 (s, 9H), 1.66-1.78 (m, 2H), 3.10-3.34 (m, 2H), 3.88 (t, 2H,
J ) 6.5 Hz), 5.01 (s, 2H), 5.61 (d, 1H, J ) 7.1 Hz), 5.90 (t, 1H,
J ) 5.5 Hz), 6.82 (d, 2H, J ) 9.2 Hz), 6.89 (d, 2H, J ) 9.2 Hz),
7.27-7.47 (m, 10H), 9.38 (d, 1H, J ) 7.1 Hz), 11.34 (s, 1H); 13
C
NMR (CDCl3) δ 25.9, 26.5, 27.9, 28.2, 29.1, 29.2, 29.3 (2C), 39.7,
58.0, 68.4, 70.6, 115.3, 115.7, 127.4, 127.5, 127.8, 128.3, 128.4,
128.8, 137.2, 137.3, 152.6, 152.7, 153.4, 155.2, 163.1, 169.4; IR
(neat) 3314, 3245, 3162, 1653, 161 cm-1; MS (ES) m/z 703 (M +
H); Anal. (C40H54N4O7) C, H, N.
1-(4-Benzyloxyphenoxy)-8-(2-N,N′-bis-tert-butoxycarbon-
ylguanidino-2-phenyl-1-ethyloxy)octane (12d). Compound 12d
(0.16 g, 79%) was prepared using a procedure similar to that for
12a: 1H NMR (CDCl3) δ 1.19-1.37 (m, 8H), 1.37-1.60 (m, 2H),
1.45 (s, 9H), 1.49 (s, 9H), 1.73 (p, 2H, J ) 6.6 Hz), 3.32-3.48
(m, 2H), 3.61-3.75 (m, 2H), 3.87 (t, 2H, J ) 6.6 Hz), 4.99 (s,
2H), 5.42-5.51 (m, 1H), 6.81 (d, 2H, J ) 9.2 Hz), 6.88 (d, 2H, J
) 9.2 Hz), 7.19-7.44 (m, 11H), 9.13 (d, 1H, J ) 8.5 Hz), 11.55
(s, 1H); 13C NMR (CDCl3) δ 25.8, 25.9, 27.9, 28.2, 29.2, 29.4,

2620 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Velu et al.
53.2, 68.3, 70.5, 70.8, 71.3, 73.2, 78.9, 82.8, 115.2, 115.6, 126.8,
127.1, 127.3, 127.7, 128.2, 128.4, 137.2, 139.7, 152.7, 152.9, 153.3,
155.6, 163.5; IR (neat) 3325, 3279 cm-1; MS (ES) m/z 690 (M +
H); Anal. (C40H55N3O7) C, H, N.
References
(1) (a) Marty, A. M. History of the development and use of biological
weapons. Clinics Lab. Med. 2001, 21, 421-434. (b) Marty, A. M.;
Conran, R. M.; Kortepeter, M. G. Recent challenges in infectious
diseases. Biological pathogens as weapons and emerging endemic
threats. Clinics Lab. Med. 2001, 21, 411-420 and references cited
therein. (c) Polgreen, P. M.; Helms, C. Vaccines, biological warfare,
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(2) (a) Singh, M. P.; Peterson, P. J.; Weiss, W. J.; Kong, F.; Greenstein,
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N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-guanidino-3-phenyl-
propionamide (13a). To a solution of compound 12a (8.21 g, 11.4
mmol) in anhydrous CH2Cl2 (50 mL), a solution of TFA (50 mL)
in anhydrous CH2Cl2 (50 mL) was added dropwise and the mixture
stirred at room temperature under N2 for 3 h. The solvent and TFA
were completely removed under vacuum. The residue was dissolved
in CH2Cl2 (100 mL), washed with saturated Na2CO3 (3 × 50 mL),
water (2 × 50 mL), and brine (50 mL), and dried over Na2SO4.
After filtering, the solvent was completely removed to obtain the
crude product. It was purified by column chromatography over Si
gel (7 × 20 cm) using MeOH/CHCl3 (1:19) as eluent to afford the
guanidine 13a (4.60 g, 78.2%). Guanidine 13a was dissolved in
MeOH (50 mL), 2 N HCl (10 mL) was added, and the mixture
was stirred for 3 h. The solvent and water were completely removed
under vacuum, and the residue was coevaporated with anhydrous
benzene (3 × 50 mL) to remove the last traces of water. Finally,
hexanes were added and the suspension was stirred and filtered to
obtain the hydrochloride salt of guanidine 13a (4.10 g, 82.9%):
1H NMR (MeOD-d4) δ 1.19-1.51 (m, 10H), 1.65-1.77 (m, 2H),
2.93-3.25 (m, 4H), 3.89 (t, 2H, J ) 6.2 Hz), 4.31 (t, 1H, J ) 8.7
Hz), 4.99 (s, 2H), 6.80 (d, 2H, J ) 9.2 Hz), 7.89 (d, 2H, J ) 9.2
Hz), 7.19-7.42 (m, 10H); MS (ES) m/z 517 (M + H); Anal.
(C31H41ClN4O3‚0.5H2O) C, H, N.
1-(4-Benzyloxyphenoxy)-8-(2-guanidino-3-phenyl-1-propyloxy)-
octane (13b). Compound 13b (0.082 g, 72%) was prepared using
a procedure similar to that for 13a: 1H NMR (CDCl3) δ 1.17-
1.45 (m, 8H), 1.45-1.61 (m, 2H), 1.72 (p, 2H, J ) 6.5 Hz), 2.73-
2.87 (m, 1H), 2.87-3.04 (m, 1H), 3.25-3.43 (m, 3H), 3.43-3.56
(m, 1H), 3.65-3.80 (m, 1H), 3.85 (t, 2H, J ) 6.5 Hz), 4.97 (s,
2H), 6.81 (d, 2H, J ) 9.2 Hz), 6.87 (d, 2H, J ) 9.2 Hz), 6.90-
7.16 (m, 1H, NH), 7.16-7.49 (m, 12H including NH2), 8.09 (d,
1H, J ) 6.5 Hz); 13C NMR (CDCl3) δ 25.6, 25.7, 29.0 (2C), 29.1
(2C), 37.1, 55.1, 68.2, 70.3, 70.1, 73.9, 115.1, 115.5, 126.8, 127.2,
127.6, 128.2, 128.5, 128.8, 136.4, 137.1, 152.5, 153.2, 158.7; IR
(neat) 3329, 3259, 3155, 1668 cm-1; MS (ES) 504 (M + H); Anal.
(C33H42F3N3O5) (analyzed as TFA salt) C, H, N.
N-[8-(4-Benzyloxyphenoxy)-1-octyl]-2-guanidino-2-phenyl-
acetamide (13c). Compound 13c (0.101 g, 88.4%) was prepared
using a procedure similar to that for 13a: 1H NMR (CDCl3) δ
0.98-1.46 (m, 10H), 1.55-1.75 (m, 2H), 2.75-3.21 (m, 2H), 3.81
(t, 2H, J ) 6.4 Hz), 4.95 (s, 2H), 5.93 (br s, 1H, NH), 6.77 (d, 2H,
J ) 9.1 Hz), 6.85 (d, 2H, J ) 9.1 Hz), 7.13-7.42 (m, 10H), 7.42-
7.56 (m, 2H, NH2), 7.97 (br s 1H, NH), 8.32 (br s, 1H, NH); 13C
NMR (CDCl3) δ 25.9, 26.6, 28.7, 29.1, 29.2, 29.3, 29.6, 40.0, 68.4,
70.5, 115.3, 115.7, 126.6, 127.4, 127.8, 128.4, 128.8, 129.1, 135.7,
137.2, 152.7, 153.3, 156.7, 169.6; IR (neat) 3314, 3245, 3162, 1653,
1611 cm-1; MS (ES) m/z 503 (M + H); Anal. (C30H39ClN4O3) C,
H, N.
1-(4-Benzyloxyphenoxy)-8-(2-guanidino-2-phenyl-1-ethyloxy)-
octane (13d). Compound 13d (0.078 g, 87.9%) was prepared using
a procedure similar to that for 13a: 1H NMR (CDCl3) δ 1.21-
1.49 (m, 8H), 1.49-1.62 (m, 2H), 1.73 (p, 2H, J ) 6.6 Hz), 3.34-
3.5 (m, 2H), 3.54-3.68 (m, 2H), 3.88 (t, 2H, J ) 6.5 Hz), 4.45-
4.57 (m, 1H), 4.99 (s, 2H), 6.82 (d, 2H, J ) 9.1 Hz), 6.89 (d, 2H,
J ) 9.1 Hz), 7.27-7.45 (m, 10H), 7.45-7.80 (br s, 3H), NH, NH2),
8.32 (d, 1H, J ) 5.6 Hz, NH); 13C NMR (CDCl3) δ 25.8, 25.9,
29.23, 29.26, 29.3, 57.8, 68.4, 70.5, 71.8, 75.3, 115.3, 115.7, 126.7,
127.4, 127.8, 128.4, 128.7, 129.1, 136.1, 137.2, 152.7, 153.4, 158.7;
IR (neat) 3318, 3261, 3147, 1664 cm-1; MS (ES) 490 (M + H);
Anal. (C30H40ClN3O3‚0.5H2O) C, H, N.
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Supporting Information Available: A table of elemental
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