Discovery of the development candidate N-tert-butyl nodulisporamide: A safe and efficacious once monthly oral agent for the control of fleas and ticks on companion animals

Article abstract of DOI:10.1021/jm801334v

Nodulisporic acid A (1) is a structurally complex fungal metabolite that exhibits systemic efficacy against fleas via modulation of an invertebrate specific glutamate-gated ion channel. In order to identify a nodulisporamide suitable for monthly oral dosi

Full text of DOI:10.1021/jm801334v

J. Med. Chem. 2009, 52, 3505–3515
3505
Discovery of the Development Candidate N-tert-Butyl Nodulisporamide: A Safe and Efficacious
Once Monthly Oral Agent for the Control of Fleas and Ticks on Companion Animals†
Peter T. Meinke,* Steven L. Colletti, Michael H. Fisher, Matthew J. Wyvratt, Thomas L. Shih,‡ Michelle B. Ayer, Chunshi Li,
Julie Lim, Dong Ok, Steve Salva, Lynn M. Warmke, Michelle Zakson, Bruce F. Michael, Pierre deMontigny, Dan A. Ostlind,
David Fink, Marlene Drag, Dennis M. Schmatz, and Wesley L. Shoop§
Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065-0900
ReceiVed October 22, 2008
Nodulisporic acid A (1) is a structurally complex fungal metabolite that exhibits systemic efficacy against
fleas via modulation of an invertebrate specific glutamate-gated ion channel. In order to identify a
nodulisporamide suitable for monthly oral dosing in dogs, a library of 335 nodulisporamides was examined
in an artificial flea feeding system for intrinsic systemic potency as well as in a mouse/bedbug assay for
systemic efficacy and safety. A cohort of 66 nodulisporamides were selected for evaluation in a dog/flea
model; pharmacokinetic analysis correlated plasma levels with flea efficacy. These efforts resulted in the
identification of the development candidate N-tert-butyl nodulisporamide (3) as a potent and efficacious
once monthly oral agent for the control of fleas and ticks on dogs and cats which was directly compared to
the topical agents fipronil and imidacloprid, with favorable results obtained. Multidose studies over 3 months
confirmed the in vivo ectoparasiticidal efficacy and established that 3 lacked overt mammalian toxicity.
Tissue distribution studies in mice using [14C]-labeled 3 indicate that adipose beds serve as ligand depots,
contributing to the long terminal half-lives of these compounds.
Introduction
imidacloprid.9 Imidacloprid is a fast acting, topical spot-on agent
which controls fleas for 1 month but lacks acaracidal activity.
Fipronil, also topically applied, is more effective, controlling
not only fleas for up to 3 months but also ticks for 4 weeks.
Infestations of blood sucking ectoparasites, such as fleas and
ticks, have long plagued both humans and animals. To alleviate
the morbidity and discomfort associated with these infestations,
considerable resources have been directed at identifying safe
and effective cidal agents.1 In part, this focus reflects a need
associated with prodigious levels of pet ownership; the American
Veterinary Medical Association currently estimates that 2007
U.S. pet ownership exceeds 71 million dogs and 81 million cats,
a substantial increase from 61 and 70 million dogs and cats,
respectively, in 2001.2 Over the years, a remarkable pharma-
copeia of topical agents (mostly flea treatments) or to a much
lesser extent oral agents (to date, with flea control only) were
developed to meet these needs; these substances are extensively
detailed elsewhere.3-6 As blood feeding ectoparasites are
efficient vectors of disease transmission (e.g., Lyme disease,
ehrlichiosis, Rocky Mountain spotted fever, etc.) and sources
of human allergens, effectively reducing flea and tick popula-
tions will confer ancillary benefits for humans.
With respect to comparably efficacious and nontoxic oral and
systemically active ectoparasiticides, the record is considerably
more modest, as long-acting oral cidal agents with suitable
efficacy and commensurate safety profiles remain elusive despite
a preference among some consumers for orally administered
agents.6 In principal, the dosing afforded by orally ingested
agents should ensure uniform coverage over a pet’s body and
its efficacy should not be mitigated by environmental exposure
to rain or bathing while also eliminating the potential for contact
exposure-based liabilities to humans. Ideally, an oral agent also
should confer immediate results, expeditiously killing both fleas
and ticks, thereby protecting against new infestations by
preventing oviposition or shedding of larvae, with reduced
propensity for flea or tick allergic dermatitis as sequelae. These
requirements, coupled with concerns associated with the po-
tential for resistance development to existing agents and the
desire to identify novel agents with benign mammalian and
environmental profiles, further spur drug discovery efforts. In
late 2007, however, a preliminary report appeared describing a
promising new systemically active pulicide.10 Spinosad, a
mixture of spinosyns A and D, is a systemically active cidal
agent with rapid onset of activity and the ability to confer 1
month’s protection against fleas only (no tick claims) following
a single oral dose.
In the context of the search for products with durable
antiparasitic efficacy and strong potential for mammalian or
environmental safety and to mitigate against the development
of resistance, identification of novel antiparasitic agents con-
tinues unabated. While multiple topical ectoparasiticidal agents
are available, including selamectin,7,8 market share is currently
governed by two main groups of ectoparasiticides: fipronil and
†
This manuscript is dedicated to the memory of Dr. Michael H. Fisher
The systemic ectoparasiticide activity of a natural product
was discovered in a Cimex lectularius mouse assay.31 Subse-
quently, a remarkable and structurally complex natural product
was identified as nodulisporic acid A11 (1, NsA A, Figure 1, R
) OH), produced by the endophytic fungus Nodulisporium sp.
(MF5954). It has the potential to usher in a new era in the
treatment of ectoparasitic infestations for companion animals.12
This fungal metabolite, despite close structural relationships to
(1926-2005).
* To whom correspondence should be addressed. Phone: +1-732-594-
3966. Fax: +1-732-594-9556. E-mail: peter_meinke@merck.com.
‡
Current address: J-Star Research, Inc., 3001 Hadley Road No. 1, South
Plainfield, NJ 07080.
§
Current address: DuPont Stine-Haskell Research Center, 1090 Elkton
Road, Newark, Delaware 19711.
a Abbreviations: BOP, benzotriazol-1-yloxytris(dimethylamino)phos-
phonium hexafluorophosphate; HOBT, N-1-hydroxybenzotriazole; HPLC,
high pressure liquid chromatography; PK, pharmacokinetics; po, per os.
10.1021/jm801334v CCC: $40.75
2009 American Chemical Society
Published on Web 05/15/2009

3506 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Meinke et al.
potent, systemic flea and tick cidal efficacy suitable for once
monthly oral dosing. To satisfy key criteria for a once monthly
agent using a clinically meaningful dosage, new NsA derivatives
initially were evaluated for intrinsic systemic activity against
the flea in vitro, then screened in a murine model using a blood
sucking ectoparasite (bedbug) prior to testing suitable derivatives
in dogs (against fleas and ticks) and in cats (against fleas only).
Results and Discussion
The animals were cared for in accordance with federal animal
welfare regulations, and the studies were approved by the
respective institutional animal care and use committees. All dogs
and cats used in the studies described herein were in excellent
health at the beginning of each study, and at no instance did
any dog or cat show any sign of adverse reaction during the
studies described herein.
A previously published flea efficacy study in dogs established
that while N-(4-methoxy)benzyl nodulisporamide (2, Table 2)
fully protected against flea infestations for approximately 2
weeks, it maintained residual efficacy for an additional 3
weeks.14 Therefore, a 9 week flea efficacy study in dogs using
2 was initiated, and the goal was to compare the effect of a
single higher dosage (30 mg/kg poa) on flea control versus two
consecutive lower dosages (15 mg/kg po) punctuated by 28 days.
The results of this pilot study are shown in Table 1 and Figure
1; efficacy was determined by challenging the dogs with 100
live fleas at various time points followed by removal and
quantitation of any live fleas 48 h postchallenge. Consistent flea
challenges were noted for the two vehicle-treated dogs held
throughout the 56 day observation period. For the 15 mg/kg po
dosing arm, the flea control response replicated with good
fidelity the earlier report by Shoop et al.14 More specifically, 2
conferred 100% flea control for the first 2 weeks after initial
(or second) dosing, followed by progressively diminishing
efficacy at weeks 3 and 4 after dosing; this efficacy profile was
reproduced following the second oral dose as well. Minimal
evidence of drug accumulation based on either flea efficacy or
plasma pharmacokinetic analysis was detected following the
second monthly dose. For the dog treated at 30 mg/kg po,
statistically significant reductions relative to control occurred
through week 5, although 100% control was only noted for the
first 14 days with breakthrough commencing at day 21. At weeks
6 and 7 (days 42 and 49), some residual control of fleas was
seen followed by complete loss of ectoparasite control at week
8. Simple modeling of pharmacokinetic disposition curves
suggested that a projected monthly dose of 2 would be
approximately 200 mg/kg po, a likely economically untenable
dosage. Clearly, identification of an analogue with improved
intrinsic potency and longer duration of action was integral to
program success.
A modular program to prepare a library of nodulisporamides
from which to identify an analogue suitable for once monthly
dosing therefore was initiated. Briefly, newly prepared deriva-
tives initially were evaluated for intrinsic systemic efficacy in
the cat flea, Ctenocephalides felis (EC50 for lethality) in an
artificial membrane in vitro feeding system.29 Ultimately,
approximately 335 newly prepared nodulisoramides were titrated
in this assay against fleas. This artificial membrane feeding assay
identified multiple analogues with useful intrinsic systemic
efficacy comparable or superior to that observed for the parent
natural product, NsA A. As evaluation of all potent compounds
in dogs for systemic flea efficacy was impractical, a murine
model for in vivo systemic activity was instituted as an
intermediate screening model, reducing compound requirements
Figure 1. Plasma disposition profile and flea efficacy (percent control)
of nodulisporamide 2 in dogs.
a large family of tremorgenic indole diterpenes,13 exhibits unique
properties: potent systemic efficacy against fleas on dogs with
rapid onset of action while devoid of acute mammalian
toxicity.14 NsA A is the preeminent member of a larger cohort
with multiple NsA fermentation congeners identified;15-18 these
natural products have broad worldwide distribution.19 To date,
none has shown biologically superior characteristics to those
possessed by NsA A itself.
Mechanism of action investigations established that this indole
diterpenoid modulates the function of a subset of the channels
targeted by agents such as ivermectin: more specifically,
invertebrate-specific glutamate-gated chloride ion channels.20
These channels, for which no mammalian homologue exists,
are vital to neurotransmission in insects, and their disruption
results in paralysis, thereby providing a biological basis for NsA
A’s potent invertebrate toxicity without concomitant vertebrate
liabilities. No activity of NsA A on ligand gated channels
(GABA, kainite, glycine) or Na and K voltage gated channels
was observed. These discoveries were of critical importance for
any systemically long-acting agent, as plasma drug exposures
will likely be considerable.
Because of their considerable structural complexity, this
indole diterpene class of antiparasitic agents largely has resisted
efforts at total synthesis. To date, only a diminutive member of
the nodulisporanes, (+)-NsA F, has succumbed to the elegant
and pioneering efforts of the Smith group,21 extending their prior
notable achievements on closely related indole alkaloids,
including paspalline22 and penetrum D.23 Prodigious efforts
directed toward total syntheses of more complex NsA family
members continue to emerge unabated from the Smith
laboratories.24,25
Preliminary medicinal chemistry efforts delineated that the
core of NsA poorly tolerated structural modification, whereas
dienoic acid side chain manipulations yielded derivatives (e.g.,
2) with improved in vitro26 and in vivo efficacy.27 Further
research established that incorporating heterocyclic surrogates
for the 3′′,4′′-olefin produced analogues that exhibited prolonged
systemic activity against both fleas and ticks on dogs.28 Despite
these attractive qualities, however, simple economic imperatives
due to synthetic complexity associated with heterocycle incor-
poration precluded the ready advancement of these derivatives
into clinical development.
These present studies were directed toward the identification
of a safe, economically viable nodulisporamide derivative with

DiscoVery of N-tert-Butyl Nodulisporamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3507
Table 1. Flea Efficacy of N-(4-Methoxy)benzyl Nodulisporamide (2) in Dogs at 15 or 30 mg/kg po
no. of live fleas recovereda at indicated week postdose
compd
dog no.
dose (mpk)
1
2
3
4
5
6
7
8
vehicle
vehicle
2
2
1
2
3
4
0
0
48
31
0
51
71
0
56
53
4
73
71
11
24
56
51
12
0
31
46
24
0
37
62
29
8
44
65
40
31
30b
15c
0
0
13
a Counts were done blindly. b Dosed at 30 mg/kg po on day 1. c Dosed at 15 mg/kg po on day 1 and day 28.
Table 2. Nodulisporamide Structures and Biological Activity Profiles
ing the potential for identifying a once monthly agent was viable.
These preliminary results established that the optimal com-
pounds bore small, aliphatic substituents on the 5′′-amide. A
combination of in vitro potency, in vivo efficacy, and terminal
half-lives in dogs served to winnow the list of 66 nodulispora-
mides to more tractable cohorts that were resynthesized for head-
to-head evaluation in dogs against fleas (C. felis) and ticks
(Dermacentor Variabilis) and in cats against fleas (C. felis) at
a potentially economically viable once monthly use dose (10
mg/kg po); these promising amide analogues (3-14) are shown
in Table 2.
bedbug
entry
R group
flea (LC50, ppm) (ED50, mg/kg)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
-OH
0.7
0.1
0.1
0.1
1.0
1.0
0.1
0.1
1.0
1.0
1.0
1.0
1.0
0.1
1.0
0.5
0.125
0.5
0.5
1.0
0.5
1.0
0.25
0.5
1.0
-NHCH2Ph(4-OCH3)
-NHC(CH3)3
-NHC(CH3)2CC≡H
-NHCH(CH3)2
-NHCH2C(CH3))CH2
-NHCH2CF3
-NHCH2CH(CH3)2
-NHC(CH3)2CO2CH3
-NHC(CH3)2C(O)CH3
-NHCH2-cyclopropyl
-NHCH2CH2CF3
-NHC(CH3)2C(OH)(CH3)2
-NHCH(CH2F)2
A dog flea and tick efficacy study of 8 weeks duration using
12 nodulisporamides (3-14) was initiated; all compounds were
dosed once at a single oral dose of 10 mg/kg po (Table 3).
Weekly flea challenges were continued until efficacy dropped
below 60%. Again in this study, consistent flea challenges in
the control dogs were observed. Repeated flea challenges (100
live unfed fleas/challenge) were made on the fifth day of each
week postdosing with flea combing occurring 48 h later on the
seventh day of each week for the 8 weeks of the study; an
additional flea challenge was made on the 29th day following
dosing with flea combing occurring on the 31st day postdosing.
Excluding nodulisporamides 7 and 11, for the first 2 weeks of
this study, 100% efficacy against fleas was noted. By week 3,
amides 10, 11, and 13 were exhibiting decreased effectiveness
in their ability to control flea infestations with 7, 9, and 14
showing breakthrough in one dog each. At week 4, for
compounds 7, 9, 10, 11, 13, and 14, at least one dog from each
pairing was showing substantial lack of flea efficacy. No
monthly flea challenges were made for dogs for which signifi-
cant loss of flea efficacy was observed at week 4 (e.g., 9, 10,
11, 13, and 14); one last confirmatory challenge was made at
week 5 for each of these compounds, then further testing on
these animals was eliminated. In general, an initial break in
coverage for a given analogue was inevitably followed by a
more precipitous loss of ectoparasite control in subsequent
weeks. Remarkably for three derivatives (3, 4, and 6), at both
1 month and 5 weeks postdose, at least one dog was noted with
100% control of fleas. However, ectoparasite control for 12
failed at week 5 and at week 7 for 4. Further examination of
the data from weeks 6 and 7 established the clear superiority
of 3 in controlling fleas with minimal breakthrough for 7 weeks
postdose. Remarkably, only at week 8 was a significant loss of
coverage observed for compound 3.
0.5
and simultaneously minimizing assay cycle times. Analogues
with suitably useful activity in the artificial membrane flea assay
subsequently were screened using a rodent model against a blood
sucking ectoparasite: the common bedbug, Cimex lectularis.31
The C. lectularis assay served to identify not only intrinsic
systemic efficacy (ED50 for efficacy) but also functioned as a
potential preliminary filter for overt mammalian toxicity (none
was observed).
Of the 335 analogues thus tested, approximately 66 semi-
synthetic analogues met the selection criteria of appropriate
activity in both models (flea LC50 e 1.0 ppm, bedbug ED50
e
1.0 mg/kg) to merit resynthesis for flea efficacy studies and
pharmacokinetic analysis in dogs. Flea systemic efficacy assay
results, C. lectularis mouse assay data, and flea efficacy and
half-life in dogs are provided for these 66 nodulisporamides
(Supporting Information, Tables I-IV). Critically, testing
numerous new nodulisporamides in dogs at potential monthly
use levels was impractical because of multiple factors, including
long in vivo assay cycle times as established for 2 (vide supra)
and, equally significantly, because of severe limitations in parent
NsA A availability. As a consequence, novel nodulisporamides
initially were screened in dogs at 5 mg/kg po, and as superior
analogues were identified, subsequent analogues were bench-
marked at 2.5 mg/kg po dosages, both to spare material and
further mitigate the long, requisite in vivo assay cycle time.
These studies confirmed prior observations that the plasma levels
of compound required for flea activity correlated well with
intrinsic flea systemic activity and that plasma half-lives of the
new compounds differed substantially from one another, indicat-
Simultaneously, in the above dog 8-week flea efficacy study
using nodulisporamides 3-14, the ability to also control
Dermacentor Variabilis ticks was probed using weekly chal-
lenges (50 ticks per challenge) for the first 31 days. Immediately
following dosing of the dogs with 3-14, the first tick challenge
was performed, with comb-counts performed 72 h later.
Cognizant of typical feeding behavior for D. Variabilis ticks,
where blood meal ingestion routinely initiates by day 3 and
terminates after engorgement on days 7-10 (Table 4), the comb-
counts were made 72 h postchallenge. Because of concerns
associated with potential for dog flea and/or tick allergy
Figure 2. Permissive and nonpermissive regions of nodulisporic
acid.

3508 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Meinke et al.
Table 3. Comparison of 12 Nodulisporamides at 10 mg/kg po in Dogs Using Fleas and Ticks
fleas on dogsb c at indicated week
ticks on dogsb d at indicated week
,
,
compd (n ) 2) T1/2 a(day) bedbug (mg/kg) flea (ppm)
1
2
3
4
M*
5
6
7
8
0
1
2
M*
vehicle
43 47 42 47 71 67 46 54 49
40 56 42 46 59 56 41 29 44
2
1
1
0
0
0
2
0
0
0
2
4
0
1
2
1
4
2
0
0
2
2
5
9
0
1
11
16
7
9
2
0
2
2
3
1
2
15
3
1
10
9
0
1
0
0
1
1
0
0
1
3
0
0
4
0
27
13
15
15
25
6
12
21
13
4
3
8.7
8.4
5.8
6.0
7.2
5.9
3.9
2.6
5.0
4.0
4.6
4.6
0.5
0.5
0.5
1.0
0.5
0.1
0.5
0.5
1.0
0.5
0.1
0.1
1.0
1.0
0.1
0.1
1.0
1.0
1.0
1.0
1.0
1.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
4
1
7
0
0
0
0
2
0
0
0
7
0
1
0
4
0
25
13
4
30 42
14
1
5
20 19 38
10 18 42
23 22 39 35
0
5
6
0
4
4
13
14
7
11
18
27
22
25 36 28 52
3
0
21
12
8
5
3
5
3
15
9
9
21
45
61
73
26
58
6
4
1
10
11
12
13
14
23 29
31 44
0
23 34
0
0
14
15
5
13
4
16
12
0
0
0
6
0
0
4
3
15 15 38
18
19
10
45
45
28
20
9
27
2
0
29 40
14
14
2
12
13
0
3
5
0
36
22
20
0
0
a Half-lives determined using days 4-21 data only; full pharmacokinetic data is provided (Supporting Information, Table V). b All counts were done
blindly. M* ) 1 month. c 100 (fleas/dog)/challenge. d 50 (ticks/dog)/challenge.
Table 4. D. Variabilis Adult Female Tick Weights Days Postchallenge
Table 5. Comparison of Flea Efficacy in Fed Cats for 12
Nodulisporamides (10 mg/kg po)
day
weight (mg)
fleas on cats at indicated weekb
0
3
10
13
a
compd (n ) 2)
T1/2 (days)
1
2
3
4
5
42
vehicle
53
56
0
1
0
0
1
0
6
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
56
61
2
25
12
9
30
52
0
40
36
19
79
60
43
7-10
680
3
6.6
3.2
4.4
2.6
6.2
2.2
4.2
3.2
4.0
4.3
3.8
7.6
37
44
12
30
54
47
45
34
39
48
61
32
43
31
60
18
58
17
20
8
dermatitis, weeks 3 and 4 tick challenges were not performed.
As the data in Table 3 illustrate, excluding the day 31 (M*)
comb-count, recovery of live ticks on control dogs was modest
relative to the numbers used in each challenge for reasons that
remain unclear. Tick challenges for 9-11, 13, and 14 were
terminated because of loss of flea control; the remaining test
compounds displayed full tick efficacy at 2 weeks but failed to
control the ticks at the end of the study.
4
5
6
9
6
60
65
17
16
45
45
0
0
0
0
3
1
0
0
0
31
18
37
7
8
A parallel flea efficacy study in cats of 4 weeks duration also
was performed using nodulisporamides 3-14 (10 mg/kg po,
100 fleas per challenge, Table 5). These assays are more
challenging, as cats typically exhibit superior grooming char-
acteristics relative to dogs. All cats were in good health at the
beginning of the study, and no adverse effects in any animal
were noted at any juncture throughout the course of the study.
Consistent flea counts were noted in the control cats; the lower
numbers of recovered fleas reflect the superior grooming talents
of cats relative to dogs. As in the previous dog studies, flea
challenges were discontinued for analogues when loss of efficacy
was noted. Generally speaking, nodulisporamides 3-14 mani-
fested lesser parasite control in cats, yielding results rather
distinct from prior dog studies. While all compounds exhibited
robust flea control at week 1, several derivatives (6, 7, 8, 14)
lost appreciable efficacy at week 2, and excluding nodulispora-
mide 3, essentially all failed to control fleas at week 3.
Interestingly, nodulisporamide 3, while in one animal efficacy
was modest at week 2 and completely abated by week 3, the
second cat showed 100% control of fleas to week 3. Little
efficacy remained at week 4 for this derivative in this cat.
9
10
11
12
13
14
87
41
25
72
16
20
47
a Half-lives determined using days 4-21 data only; full pharmacokinetic
data is provided (Supporting Information, Table VI). b All counts were done
blindly. 100 (fleas/cat)/challenge.
On the basis of the flea efficacy results from the head-to-
head study of compounds 3-14 in dogs, clearly nodulispora-
mides 3, 4, 8, and 12 were among the most efficacious agents.
Incorporating the tick efficacy results from the same study failed
to winnow the list further, as all but 10 and 13 had attractive
tick activity at week 2. In addition, with the absence of
potentially discriminating weeks 3 and 4 tick challenges, a broad

DiscoVery of N-tert-Butyl Nodulisporamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3509
week 9 did loss of efficacy approach 50%. For the lower dosage
of 3, 100% and 98% flea control was noted at weeks 3 and 4,
respectively. By weeks 5 and 6, this dosing arm produced
diminished efficacy. The pharmacokinetic analysis of 3 from
this study was consistent with the observation that excellent
ectoparasite control is maintained until systemic plasma drug
levels decrease to approximately e0.20 µg/mL. Large numbers
of dead fleas were combed from the dogs in the first month of
this study for both dosing arms of 3 and up to weeks 7 and 8
for the 10 mg/kg po arm, whereas all fleas from control dogs
were viable. In addition, the hair coats of these treated dogs
were largely free of flea feces until weeks 7 and 8 for this higher
dosage. Only in the ninth week did fleas on these treated dogs
begin to approach the size of those on control dogs, did females
begin to survive, and did significant levels of flea feces begin
to manifest in the dog hair coats.
Currently, fipronil and imidacloprid are the most popular
topical agents marketed for companion animals. In order to
rigorously assess the systemic ectoparasitic efficacy of 3
versus that of these two useful topical molecules, a direct
comparison was made (Table 6). Fipronil and imidacloprid
were applied as per their labeling to the backs of dogs (n )
3 per arm), using their optimized commercial formulations
at their recommended use levels. In this head-to-head study,
the efficacy profile of the orally active nodulisporamide 3
compared favorably to the efficacy profiles of both topical
agents. In this four arm study, only the 5 mg/kg po dosing
arm for 3 exhibited less than 100% flea efficacy at 1 month
postdosing. Breakthrough in parasite control occurred earliest
for imidacloprid at week 5 and progressed further in week
6. Imidacloprid exhibited 100% efficacy at 1 month (M*),
with breakthrough occurring in week 5 and further erosion
of efficacy at week 6. The topical agent fipronil, on the other
hand, controlled 100% of fleas at week 5, with >90% efficacy
persisting to the end of the study at week 9. Nodulisporamide
3, at 10 mg/kg po, conferred 100% control of fleas at week
6 and >90% flea control in weeks 7 and 8 before significant
erosion of activity manifested itself at week 9.
Figure 3. Comparison of plasma disposition profiles of 3 in dogs and
cats.
loss of efficacy by day 31 (M*) made compound selection a
challenge that was further exacerbated by the modest recovery
of live D. Variabilis ticks from control animals. Fortunately,
however, when the additional results from the corresponding
study in cats were assimilated, the aggregate data were suf-
ficiently unambiguous that of the 12 compounds profiled only
N-tert-butyl nodulisporamide (3) displayed the desired optimal
balance of ectoparasitic activity in both dogs and cats.
A comparison of the PK profiles of 3 in both cats and dogs
clarifies the origin of the reduced feline efficacy. In cats, the
shorter t1/2 (6.6 vs 8.7 days) and lower Cmax (1.52 ( 1.65 vs
9.8 ( 0.28 µM) values contribute to the observed differential
(Figure 3). It should be noted, however, that the cat for which
the greatest flea efficacy (100% at week 3) was measured was
the animal exposed to the highest drug levels of 3. Overall, the
plasma disposition curves for 3 are similar in dogs and cats. In
addition, preliminary data suggest that the oral bioavailability
in cats is similar to that of dogs (75-85%) and independent of
dose. Plasma concentrations of the compound in both species
again correlate well with observed flea efficacy with levels of
0.15-0.20 µg/mL being required for 100% flea control. Nod-
ulisporamide 3 has multicompartment pharmacokinetics, best
described with a triexponential equation, where the third
exponential rate constant represents the “apparent” elimination
phase. The first two exponential terms presumably represent a
rapid and a slow distribution sequence, suggesting that the
majority of the compound is partitioned into both highly and
poorly perfused tissues. The “apparent” elimination half-life of
3 is ∼19 days. At the proposed doses, plasma levels are low
enough at 30 days so that no significant drug accumulation is
anticipated following repeated monthly treatment. These results
also indicate that minimal metabolism occurs.
As a consequence of the above conclusion, sufficient 3 was
resynthesized to support multiple efficacy studies to more
completely profile the qualities of this remarkable material. First,
a larger confirmatory study to evaluate the efficacy of 3 against
fleas on dogs was initiated (Table 6). In this 9 week study, two
single dosages of 3 were utilized: 5 and 10 mg/kg po (n ) 4
per arm) to determine if a lower dosage would confer 1 month’s
protection against fleas and ticks. As before, no adverse effect
of 3 in the dogs was detected in this study. Again, consistent
live flea counts were seen throughout this study on the control
animals. Remarkably, following a single oral dose of 3 at 10
mg/kg po, 100% flea control was observed for 6 weeks. Partial
loss of efficacy was observed in weeks 7 and 8 and not until
In the confirmatory 9 week investigation described above,
brown dog ticks (Riphicephalus sanguineus) also were included,
as they are the most prevalent dog tick in the U.S. For each
challenge, 50 ticks were placed on each dog, and 72 h later the
dogs were combed to remove and count any remaining live ticks.
To avoid compromising the flea study, tick challenges were
interspersed between flea challenges. In this study, the 10 mg/
kg po dose of 3 was 80%, 78%, and 41% efficacious at 12, 19,
and 26 days, respectively. In contrast, fipronil was 91% and
77% effective at days 19 and 26, respectively. As imidacloprid
lacks activity against ticks, it did not merit inclusion in this
aspect of the investigation.
A corresponding dose confirmation study in cats using
nodulisporamide 3 also was performed (Table 7). In recognition
that less robust efficacy had been noted in the prior cat study,
the utility of higher dosages (specifically, 15 and 20 mg/kg po)
was probed in this larger study. No adverse event of 3 in any
cat was noted during the course of this 6-week study. Also,
consistent numbers of live fleas were recovered from the control
animals throughout the course of this 6-week study. In this
investigation, a single oral dose of 15 mg/kg po conferred 100%
efficacy for 2 weeks, with breakthrough occurring at week 3
and progressive loss of efficacy evident in weeks 4, 5, and 6.
At the higher dosage of 20 mg/kg po, however, 100% efficacy
against fleas was observed at week 3, with 97% and 94% control
at weeks 4 and 5, respectively. Only in the final, sixth week

3510 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Meinke et al.
Table 6. Dose Confirmatory Study in Dogs Using Nodulisporamide 3 against Fleas and R. sanguineus Ticks
fleas on dogsa b at indicated week postdosing
R. sanguineus ticksb c at indicated day
,
,
1
2
3
4
M*
5
6
7
8
9
3
12
19
26
Vehicle (n ) 4)
33
64
70
48
60
47
44
40
51
54
55
40
54
61
34
57
55
71
74
61
71
61
51
40
41
54
53
45
61
62
63
44
62
62
83
35
56
59
58
35
27
21
18
13
12
14
2
19
17
11
11
23
15
25
11
6
3 (10 mg/kg po) (n ) 4)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
4
0
1
0
7
1
0
0
0
0
2
3
2
0
4
0
5
4
11
10
17
6
26
15
11
3 (5 mg/kg po) (n ) 4)
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
3
1
0
0
5
5
10
1
15
12
0
0
0
0
0
8
5
8
9
29
7
6
5
7
14
Imidacloprid, Spot-On (2.5 mL, n ) 3)
21
6
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
6
4
2
3
Fipronil Spray (3.3 Pumps/kg, n ) 3)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
2
0
0
3
0
1
13
0
0
1
1
0
0
a Challenged with 100 fleas/dog 48 h prior to counting. M* ) 1 month. b All counts were performed blindly. c Challenged with 50 ticks/dog 72 h prior
to counting.
Table 7. Dose Confirmation Study in Fed Cats Using
Nodulisporamide 3
The prior tick efficacy study (Table 6) using dogs dosed at 5
and 10 mg/kg po established that higher dosages of 3 were
integral to achieve 1 month’s coverage of ticks. As a conse-
quence, a larger study was initiated using 30 mg/kg po dosages
of 3, and to further extend our understanding of the ability of
3 to control ticks, a second species, Amblyoma americanum
(Lone Star ticks), also was introduced (Table 8). In this study,
consistent numbers of both species of ticks were recovered from
the control animals. At day 14, 3 conferred 100% protection
from both species of ticks. Despite the higher dosage, break-
through was seen against both tick species at days 21 (85%
and 87% control) and 28 (84% and 92% control) for R.
sanguineus and A. americanum, respectively, suggesting that
robust tick efficacy is manifest only while plasma drug
concentrations exceed 1 µg/mL levels (Supporting Information,
Figure II). Flea efficacy also was determined for 3 at two time
points in this study: days 61 and 90 postdosing (Table 8). The
observed flea control was 100% and 23% at these respective
time points. Systemic plasma exposures of 3 were ∼0.17 µM
on day 61 and ∼0.05 µM on day 90, establishing that a single
oral dose of 3 can confer >60 days, but not 90 days, of full
protection against fleas (Supporting Information, Figure II).
fleas on catsa at indicated week postdosing
1
2
3
4
5
6
Vehicle (n ) 4)
26
31
36
52
20
38
44
43
27
37
39
56
34
36
37
51
31
39
53
38
20
29
29
60
3 (15 mg/kg po) (n ) 4)
0
0
0
0
0
0
0
0
1
1
0
5
14
1
2
15
6
10
18
14
15
41
6
19
3 (20 mg/kg po) (n ) 4)
0
0
0
0
0
0
0
0
0
0
0
0
1
2
1
0
0
0
4
5
10
3
6
14
Imidacloprid Spot-On (n ) 3)
0
0
0
1
0
0
0
0
0
1
0
1
2
1
0
0
1
1
Blood Sampling and Plasma Analysis. A significant concern
with compounds with long half-lives is that the potential for
accumulation subsequent to multiple dosing exists. Shown below
in Figure 4 is the pharmacokinetic profile of nodulisporamide
3 dosed at 10 mg/kg po for 4 consecutive months. No flea
challenges were performed in this study to minimize flea allergic
dermatitis risk to the control animals. From this semilog plot,
it is apparent that consistent mean exposures and Cmax and Ctrough
levels were attained between animals for each dosage across
the 4 month dosing window, suggesting minimal risk of
accumulation of drug in a chronic dosing setting. Again, no
signs of behavioral changes or toxicity were noted in the animals
throughout the duration of this study.
Fipronil Spray (n ) 3)
0
0
0
0
0
0
0
0
0
1
0
0
0
5
8
1
6
16
a Challenged with 100 fleas at 48 h prior to counting. All counts were
performed blindly.
did the efficacy for 3 decrease substantially, to 76%. As with
the prior dog study (Table 6), until systemic plasma drug
exposures diminished to 0.2-0.3 µg/mL (Supporting Informa-
tion, Table X), full control of the blood-sucking fleas was
observed. Both topical agents (fipronil and imidacloprid) showed
excellent duration of action in cats, providing essentially full
efficacy against fleas at week 4, with relatively modest loss of
efficacy detected at weeks 5 and 6 for imidacloprid and more
substantial breakthrough in flea control observed for fipronil.
As part of the routine characterization of potential develop-
ment candidates, nodulisporamide 3 was tested in a battery of

DiscoVery of N-tert-Butyl Nodulisporamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3511
Table 8. Efficacy of Nodulisporamide 3 in Dogs Orally at 30 mg/kg po against Fleas and Ticks
R. sanguineusa b on indicated day
A. americanuma b on indicated day
fleasb c on indicated day
,
,
,
3
7
14
21
28
32
3
7
14
21
28
32
61
90
Vehicle (n ) 4)
27
21
20
27
10
27
14
25
10
14
15
11
18
16
20
26
20
25
23
35
20
11
27
23
24
36
29
26
25
29
25
20
22
26
24
16
30
39
44
46
25
22
26
20
33
69
80
77
81
79
72
90
90
27
28
11
3 (n ) 4)
0
0
0
0
0
0
0
0
0
0
0
0
3
0
3
6
2
5
6
3
7
4
8
9
0
0
0
0
0
0
0
0
0
0
0
0
4
2
3
3
2
8
3
0
7
12
20
33
0
0
0
0
77
63
96
34
a Challenged with 50 ticks of each species per dog at 72 h prior to counting. b All counts were performed blindly. c Challenged with 100 fleas per dog
48 h prior to counting.
Figure 5. Tissue distribution of [14C]-N-tert-butyl nodulisporamide
(3) in mice.
Figure 4. Pharmacokinetic profile of 3: multiple monthly dosing at
10 mg/kg po (91 day study).
distribution of [14C]3 is distinct from that seen at prior time
points. The histogram indicates that a significant depot effect
assays to better understand the qualities and limitations of the
molecule. Compound 3 was devoid of meaningful activity at
10 µM in a Panlabs panel of 102 in vitro assays with the
exception of 90% inhibition of monoamine uptake at 10 µM
of [14C]3 occurs over time with maximal drug absorbing into
abdominal and altilateral adipose tissue. Presumably, [14C]3
gradually leaches out of these adipose depot beds, contributing
to the prolonged terminal half-life observed for 3 and its
extensive duration of efficacy against ectoparasites.
and 52% inhibition at 1 µM. No inhibition of dog P450 enzymes
was noted at 10 µM. Nodulisporamide 3 was radiolabeled by
reacting H2N14C(CH3)3 with the previously isolated and purified
Chemistry. Nodulisporamides 2-14 were derived in a single
HOBT ester of nodulisporic acid A (Scheme 1). Covalent
chemical step from nodulisporic acid A (1) in coupling yields
binding studies using [14C]3 in rat and dog liver microsomes
ranging from 35% to 70% (Scheme 1).26 Unsurprisingly, the
were unremarkable, yielding <50 pM equiv/mg protein labeling
observed chemical yield was significantly affected by the purity
in the presence or absence of NADPH, GSH (5 mM), or NaCN
(1 mM). Nodulisporamide 3 is highly plasma protein bound
of the starting carboxylic acid, which typically ranged from 80%
to 90%. If the NsA A was relatively pure, the corresponding
(99.3%), which also may contribute to both its excellent
nodulisporamides were readily obtained following flash chro-
mammalian safety profile and serve as the drug delivery vehicle
matography on silica gel. Often, however, 1 was heavily
contaminated with multiple fermentation congeners (including
2′-epi-NsA A, 3′′,4′′-Z-NsA A, nodulisporic acids B, C, D, E,
and F), raising purification challenges. In those instances, both
preparative normal phase and reversed phase HPLC purification
were necessary to obtain pure nodulisporamides. Alternatively,
if desired, the intermediate NsA HOBT ester could be isolated
by column or thin layer chromatography and later be subjected
for blood sucking ectoparasites.
CD-1 mice were dosed orally with [14C]3 at 5 and 20 mg/kg.
Tissue distribution of [14C]3 was determined at 8 h, 21 days,
and 28 days postdose. Initially, at 8 h postdose, the radiolabeled
material is predominantly present in the stomach and gas-
trointestinal tract (Supporting Information, Figure I for 8 h and
5 mpk data). In contrast, at days 21 and 28 postdose, as
illustrated in Figure 5 for the 20 mg/kg dosing arm, the
Scheme 1. General Synthesis of Nodulisporamide Derivatives

3512 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Meinke et al.
randomly allocated to control or treatment groups, and the groups
were allocated randomly to treatment. All dogs were in excellent
health at the beginning of each study described below, and no dog
showed any sign of adverse reaction during these studies.
Briefly, for the trial described in Table 3, 26 beagle dogs of
similar weight (10.0-13.5 kg) were allocated randomly to 1 of 12
groups (n ) 2, one male and one female per treatment arm), and
the groups were allocated randomly to treatment. The treatment
arms were nodulisporamides 3-14 (Table 1) or vehicle treated
control. Each compound was administered per os via a calibrated
syringe at 10 mg/kg at 0.2 mL/kg on day 0. The vehicle was
dimethyl sulfoxide/propylene glycol/glycerol formal (5:3:2). Flea
challenges were made by placing 100 live, unfed fleas, Cteno-
cephalides felis, from our flea colony along the dorsal midline of
each dog on the evening before the trial. The fleas were comb-
counted 48 h after treatment according to the methods of Zakson
et al.32 Five days after the 48 h comb-count, a fresh batch of 100
unfed fleas was placed onto each dog. This weekly comb-count
iteration was continued for the duration of each assay. In two assays,
an additional flea challenge was performed on day 31 (M*), where
fleas were placed on the dogs. All counts were done in a “blind”
fashion.
to amide bond forming reactions; this ester was stable for weeks
at -20 °C under nitrogen.
Conclusion
A modular synthesis effort was utilized to prepare a library
of nodulisporamide analogues from which the development
candidate N-tert-butyl nodulisporamide (3) was identified.
Through a combination of potency and extended half-life, a
single oral dose of 3 can protect dogs (5-10 mg/kg) and cats
(15-20 mg/kg) against fleas for an entire month. In addition,
at higher dosages, 3 is 100% effective against ticks upon dosing
and retains tick activity (84-92%) throughout the month. A
higher single oral dose of 30 mg/kg in dogs may protect against
flea infestations for greater than 2 months. No commercial
product has the unique oral properties of nodulisporamide, and
its systemic efficacy profile compares favorably to currently
marketed topical agents. Mechanism of action studies provide
a biological basis for the safety profile of this systemically active
agent and potent cidal activity against blood sucking ectoparasites.
Cat Flea Efficacy Tests. Two independent cat flea efficacy
studies were performed; each trial used different numbers of animals
and had different durations but followed the general protocols
developed by Shoop et al.14 as described above using fed cats (males
only) instead of dogs. In all cases, animals were randomly allocated
to control or treatment groups, and the groups were allocated
randomly to treatment. All cats were in excellent health at the
beginning of each study, and no cat showed any sign of adverse
reaction during these studies.
Dog Tick Efficacy Studies. Three independent tick efficacy
studies were performed using three different species of ticks from
our colonies, as detailed in Table 3 [Dermacentor Variabilis
(American Dog tick)], Table 4 [Rhipicephalus sanguineus (Brown
Dog tick)], and Table 7 [R. sanguineus and Amblyoma americanum
(Lone Star tick)]. All dogs were in good health at the beginning of
each study described below, and no dog showed any sign of adverse
reaction during these studies.
For the trial described in Table 3, concurrent with the flea efficacy
study, the beagle dogs dosed with nodulisporamides 3-14 and the
vehicle treated beagles also were exposed to repeated tick chal-
lenges, interleaved at the indicated time points. Tick challenges
were made by placing 50 live, unfed D. Variabilis ticks from our
tick colony along the dorsal midline of each dog on day -2 of the
trial. The ticks were comb-counted 72 h after treatment according
to the methods of Zakson et al. previously developed for counting
fleas.32 Careful inspection of the combed parasites permitted
identification and quantification of recovered ticks. Four days
subsequent to the initial 72 h comb-count, a fresh batch of 50 unfed
D. Variabilis ticks was placed onto each dog. This weekly comb-
count iteration was continued on days 7, 14, and 31 (M*). All counts
were done in a “blind” fashion.
For the study detailed in Table 7, nodulisporamide 3 was
administered per os via a calibrated syringe at 30 mg/kg at 0.2
mL/kg on day 0 (n ) 4 male beagle dogs per treatment or vehicle-
treated control arm). The vehicle was dimethyl sulfoxide/propylene
glycol/glycerol formal (5:3:2). Only male beagle dogs were used,
and all beagle dogs were of similar weight (10.0-13.5 kg). In all
cases, animals were randomly allocated to control or treatment
groups, and the groups were allocated randomly to treatment. Tick
challenges were made by placing 50 live, unfed R. sanguineus ticks
and 50 live, unfed A. americanum ticks from our tick colonies along
the dorsal midline of each dog on day 0 of the trial. This study
was otherwise conducted exactly as described in the preceding
paragraph.
Experimental Section
Artificial Membrane Flea Feeder. Artificial membrane flea
feeding devices have been described previously.29,30 In brief, it is
a plexiglass chamber divided into a heated upper and a nonheated
lower and has a series of 104 ports (a 25 port variant is sold
commercially by Jay Georgi, FleaData, Inc., Freeville, NY). Bovine
blood spiked with various drugs and concentrations was introduced
into aluminum sleeves with PARAFILM stretched across the sleeve
bottom to form a membrane. Each blood-filled sleeve was placed
into the heated upper half with the PARAFILM membrane
interfacing with 1 of the 104 ports. A small cage with 25 adult
fleas fed on the warm bovine blood through the PARAFILM
membrane. The blood in the heated chamber was maintained at 40
°C, and the fleas in the bottom half were maintained at 28 °C and
85% relative humidity (RH).
NsA A and the nodulisporamide derivatives were dissolved in
polyethylene glycol 400 and dimethyl sulfoxide (2:1) and were
added to 10 mL of bovine blood in each aluminum sleeve to achieve
concentrations of 10.0, 1.0, and 0.1 µg/mL. The bovine blood had
been treated with citrated dextrose as an anticoagulant. After 24 h
of feeding, the sleeves were replaced with fresh blood and drug.
The fleas were allowed to feed on the fresh drug-treated blood for
another 24 h after a total of 48 h of drug exposure. The cages were
removed, and the living and dead fleas were counted. Lethal
concentrations that killed 50% of fleas (LC50) were calculated using
linear regression.
Cimex lectularis Efficacy Test. The Cimex lectularis assay has
been described previously.31 Briefly, female CD-1 mice (n ) 3)
were given compounds orally via gavage (four dose levels: 1.0,
0.5, 0.25, and 0.125 mg/kg) 24 h prior to feeding the fourth stage
instars. The single orally dosed compounds were dissolved in a
mixture of polyethylene glycol 400 and DMSO (1:2, v/v) and
administered at a rate of 0.5 mL/100 g, with concentrations adjusted
to provide the specified levels in mg/kg. Following treatment (24
h for single oral doses) the mice were restrained in plastic containers
from which their tails were put through a small hole. The tail was
washed, wiped dry, and inserted into a shell vial containing five
unfed fourth instar C. lectularius. The vials containing the engorged
insects were removed, plugged with a foam stopper, and examined
daily until 80% or more of the control instars had molted to the
fifth instar. Death, paralysis, or molt delay relative to the placebo-
treated controls served as the criteria for activity.
Dog Flea Efficacy Tests. Multiple independent dog flea efficacy
studies were performed; each trial used different numbers of animals
and had different length but followed the general protocols
developed by Shoop et al.14 With the exclusion of the trial detailed
in Table 3, only male beagle dogs were used, and all beagle dogs
were of similar weight (10.0-13.5 kg). In all cases, animals were
Blood Sampling and Plasma Analysis. Blood samples were
taken from treated dogs in heparinized VACUTAINER tubes at
various times during each study. Concentrations of the administered
compounds were determined in plasma using HPLC after analyte
extraction by protein precipitation. For the nodulisporamides, 0.5

DiscoVery of N-tert-Butyl Nodulisporamide
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3513
mL of plasma was diluted with an equal volume of water, and 3.5
mL of acetonitrile was added with gentle vortexing to precipitate
the plasma proteins. After removal of solids by centrifugation, the
supernatant liquid was reduced to dryness, and the residue was
reconstituted with 0.2 mL of 50% aqueous acetonitrile. The resulting
solution was analyzed by HPLC with ultraviolet photometric
detection using a YMC ODS-AM column (150 mm × 3 mm, 3
µm particle diameter) at 40 °C with an eluant of acetonitrile and
water (4:1). The flow rate was 0.4 mL/min, the injection volume
was 100 µL, and the detection wavelength was 380 nm.
General Procedure for the Preparation of Nodulisporic
Acid A (NsA A, 1). NsA A was fermented, isolated, and purified
according to the methods outlined by Ondeyka et al.11 Briefly, NsA
A was purified from methyl ethyl ketone extracts of Nodulisporium
sp., grown either on solid substrate or in liquid medium. The extracts
were dried and portioned between hexane and methanol (1:1), and
the methanol soluble fraction was chromatographed over silica gel,
followed by gel filtration. Final purification was achieved by
preparative reversed-phase high-performance liquid chromatography
(HPLC), which provided pure NsA A as a yellow powder.
1.47 (s, 3H), 1.38 (s, 9H), 1.34 (s, 3H), 1.32 (s, 3H), 1.12 (s, 3H),
1.11 (s, 3H), 1.03 (s, 3H), 0.93 (s, 3H). 13C NMR spectrum (125
MHz, CDCl3, ppm) δ 197.56, 168.52, 154.03, 150.21, 139.29,
137.94, 135.34, 133.62, 132.50, 130.09, 128.16, 125.46, 122.09,
121.49, 121.81, 117.42, 116.45, 112.69, 76.49, 75.95, 74.81, 73.61,
72.26, 57.54, 55.50, 51.06, 47.45, 46.94, 44.82, 38.55, 31.80, 37.74,
29.79, 29.61, 28.67, 27.34, 25.22, 24.08, 23.12, 19.24,17.74,14.95,
13.12, 11.03. IR (film, NaCl) 3348.6, 2972.8, 2936.1, 1707.6,
1655.7, 1509.7, 1509.5, 1452.9, 1378.8, 1229.8, 1075.8, 1022.0,
976.7 cm-1. UV-vis λmax (MeCN) 245, 260, 390 nm. HRMS (ESI,
pos mode) calculated for C47H62N2O5, 734.4658; found m/z )
735.473 12 [M + H]+.
N-(4-Methoxy)benzyl Nodulisporamide (2). Yield: 320 mg,
54%. TLC, silica gel: Rf ) 0.21 (1:2 acetone/hexanes). RP-HPLC
(C18 ODS, 1 mL/min, 4:1 MeCN/H2O, isocratic): tR ) 11.0 min.
1H NMR (500 MHz, CDCl3, ppm) δ 7.71 (s, 1H), 7.25 (d, J ) 8.4
Hz, 2H), 6.99 (d, J ) 10.7 Hz, 1H), 6.89 (d, J ) 8.4 Hz, 2H), 6.35
(dd, J ) 11.2, 15.4 Hz, 1H), 6.06 (d, J ) 2.7 Hz, 1H), 5.95 (t, J
) 5.5 Hz, 1H), 5.79 (d, J ) 15.6 Hz, 1H), 5.26 (d, J ) 6.2 Hz,
1H), 5.21 (s, 1H), 5.09 (s, 1H), 5.00 (br s, 1H), 4.48 (d, J ) 5.5
Hz, 2H), 3.82 (s, 3H), 3.40 (br s, 1H), 2.89 (dd, J ) 2.8, 6.2 Hz,
1H), 2.84 (br s, 1H), 2.73 (dd, J ) 6.4, 14.0 Hz, 1H), 2.32 (dd, J
) 10.9, 13.5 Hz, 1H), 2.00 (s, 3H), 1.95-1.40 (m, 11H), 1.60 (br
s, 3H), 1.51 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 1.16 (s, 3H), 1.15
(s, 3H), 1.06 (s, 3H), 0.96 (s, 3H). HRMS (ESI, pos mode)
calculated for C51H62N2O6 798.4608; found m/z ) 799.463 75
[M + H]+.
General Procedure for the Synthesis of Nodulisporamides
2-14. All 66 semisynthetic nodulisporamides, including 2-14,
were prepared by Medicinal Chemistry at Merck Research Labo-
ratories (Merck & Co., Inc., Rahway, New Jersey) as described
previously26 as illustrated for N-tert-butyl nodulisporamide (3)
below. Purity for all new nodulisporamides described herein was
determined by RP-HPLC and normal phase HPLC analysis (g97%
purity required for samples submitted for in vivo evaluation). To
NsA A (1, 2.01 g, 2.96 mmol, ∼90% pure) in 50 mL of methylene
chloride at 0 °C was added HOBT (0.20 g, 1.48 mmol), triethy-
lamine (1.30 mL, 9.27 mmol), and tert-butylamine (3.1 mL, 29.6
mL). To this solution was added benzotriazol-1-yloxytris(dimethy-
lamino)phosphonium hexafluorophosphate (BOP, 2.2 g, 4.98 mmol).
After aging for 1 h at 0 °C, the reaction mixture was partitioned
between saturated sodium bicarbonate and methylene chloride and
extracted with methylene chloride (3 × 20 mL). The combined
organic layers were washed once with saturated sodium chloride
and then dried (sodium sulfate). The solution was filtered, concen-
trated under reduced pressure, and purified by flash chromatog-
raphy on silica gel (8 cm × 15 cm column) using gradiant elution.
The silica gel was equilibrated with 25:75 EtOAc/hexanes and the
reaction crude loaded using 40 mL of methylene chloride. The
column was flushed with 25:75 EtOAc/hexanes (1 L, 3 remained
at the baseline), 33:67 EtOAc/hexanes (2 L, 3 began to elute), 40:
60 EtOAc/hexanes (until 3 began to elute off the column), and then
50:50 EtOAc/hexanes during product collection. Concentration
under reduced pressure, yielded impure 3 (1.35 g, ∼80% pure) as
a bright-yellow amorphous solid. A 6 cm × 30 cm axial compres-
sion HPLC column was slurry-packed with 20 µm silica gel. The
column was equilibrated with 25:75 EtOAc/hexanes (flow rate, 220
mL/min; λ ) 270 nm). N-1-tert-Butyl nodulisporamide (1.35 g,
68% crude yield, ∼80% pure) was loaded onto the column with
approximately 150 mL of methylene chloride. The column was
flushed using 1 L of 25:75 EtOAc/hexanes, and then the product
was eluted using 50:50 EtOAc/hexanes. Pure 3 began to elute after
20 min and was collected in 110 mL cuts. After 35 min, all 3 had
been eluted from the column. Concentration under reduced pressure
yielded pure 3 (0.85 g, 44%). Mp: 196-203 °C (decomposed).
TLC: 1:1 EtOAc/hexanes, Rf ) 0.38. HPLC (reversed-phase,
ZORBAX Rx-C8, 4.6 mm × 25 cm, 8:2 MeCN/H2O isocratic, 1.5
mL/min, λ ) 254 nm) tR ) 6.67 min. HPLC (normal phase,
ZORBAX Rx-Sil, 4.6 mm × 25 cm, 1:1 EtOAc/hexanes isocratic,
1.5 mL/min, λ ) 254 nm) tR ) 8.52 min. [R]2D0 + 17.4° (c 0.82,
CH2Cl2). log P ) 7.6 (65:35 MeOH/0.04 M phosphate buffer, 37
N-(1,1-Dimethyl)proparg-2-yl Nodulisporamide (4). Yield: 74
mg, 67%. TLC, silica gel: Rf ) 0.44, 1:1 acetone/hexanes. HPLC
(reversed-phase, ZORBAX Rx-C8, 4.6 mm × 25 cm, 6:4 to 8:2
MeCN/H2O, 20 min gradient, 2.0 mL/min, λ ) 254 nm) tR ) 22.4
min. 1H NMR (500 MHz, CDCl3, ppm) δ partial spectrum 7.67 (s,
1H), 6.90 (d, J ) 10.8 Hz, 1H), 6.30 (dd, J ) 10.9, 15.4 Hz, 1H),
6.02 (d, J ) 2.7 Hz, 1H), 5.80 (s, 1H), 5.74 (d, J ) 15.3 Hz, 1H),
5.21 (dd, J ) 2.3, 6.2 Hz, 1H), 5.17 (s, 1H), 5.05 (s, 1H), 4.96 (br
s, 1H), 3.74 (s, 1H), 3.36 (br s, 1H), 3.18 (d, J ) 2.1 Hz, 1H), 2.85
(dd, J ) 2.8, 6.2 Hz, 1H), 2.80 (m, 1H), 2.71 (dd, J ) 6.5, 13.7
Hz, 1H), 2.60 (s, 1H), 2.28 (dd, J ) 10.8, 13.8 Hz, 1H), 2.15 (s,
1H), 1.94 (s, 3H), 1.95-1.40 (m, 11H), 1.67 (s, 3H), 1.47 (s, 3H),
1.33 (s, 3H), 1.32 (s, 3H), 1.23 (s, 3H), 1.12 (s, 3H), 1.11 (s, 3H),
1.03 (s, 3H), 0.92 (s, 3H). HRMS (ESI, pos mode) calculated for
C48H60N2O5, 744.450 11; found m/z ) 745.457 39 [M + H]+.
N-Isopropyl Nodulisporamide (5). Yield: 476 mg, 60%. TLC,
1
silica gel: Rf ) 0.75 (1:9 MeOH/CH2Cl2). H NMR (500 MHz,
CDCl3, ppm) δ 7.67 (s, 1H), 6.91 (d, J ) 11.0 Hz, 1H), 6.31 (dd,
J ) 11.0, 15.1 Hz, 1H), 6.02 (d, J ) 2.8 Hz, 1H), 5.75 (d, J )
15.3 Hz, 1H), 5.51 (d, J ) 7.8 Hz, 1H), 5.22 (dd, J ) 1.7, 6.1 Hz,
1H), 5.18 (s, 1H), 5.06 (s, 1H), 4.97 (br s, 1H), 4.15 (m, 1H), 3.38
(br s, 1H), 3.16 (s, 1H), 2.85 (dd, J ) 2.8, 6.3 Hz, 1H), 2.81 (m,
1H), 2.71 (dd, J ) 6.6, 14.0 Hz, 1H), 2.27 (dd, J ) 11.1, 13.9 Hz,
1H), 1.95 (s, 3H), 1.95-1.40 (m, 10H), 1.55 (s, 3H), 1.47 (s, 3H),
1.34 (s, 3H), 1.32 (s, 3H), 1.18 (s, 3H), 1.17 (s, 3H), 1.13 (s, 3H),
1.11 (s, 3H), 1.03 (s, 3H), 0.93 (s, 3H). HRMS (ESI, pos mode)
calculated for C46H60N2O5, 720.450 57; found m/z ) 721.457 84
[M + H]+.
N-(2-Methyl)propen-2-yl Nodulisporamide (6). Yield: 470 mg,
1
54%. TLC, silica gel: Rf ) 0.72 (1:9 MeOH/CH2Cl2). H NMR
(500 MHz, CDCl3, ppm) δ 7.69 (s, 1H), 6.97 (d, J ) 11.3 Hz,
1H), 6.33 (dd, J ) 10.9, 15.0 Hz, 1H), 6.03 (d, J ) 2.9 Hz, 1H),
5.80 (m, 2H), 5.24 (d, J ) 6.3 Hz, 1H), 5.20 (s, 1H), 5.07 (s, 1H),
4.98 (br s, 1H), 4.84 (d, J ) 5.8 Hz, 1H), 3.89 (d, J ) 7.4 Hz, 2H),
3.39 (m, 1H), 3.18 (s, 1H), 2.86 (dd, J ) 2.7, 6.1 Hz, 1H), 2.80
(m, 1H), 2.70 (dd, J ) 6.3, 13.9 Hz, 1H), 2.28 (dd, J ) 10.7, 13.7
Hz, 1H), 1.99 (s, 3H), 1.95-1.40 (m, 11H), 1.74 (s, 3H), 1.54 (s,
3H), 1.45 (s, 3H), 1.36 (s, 3H), 1.34 (s, 3H), 1.13 (s, 3H), 1.11 (s,
3H), 1.03 (s, 3H), 0.93 (s, 3H). HRMS (ESI, pos mode) calculated
for C47H60N2O5, 732.4501; found m/z ) 733.457 40 [M + H]+.
N-2,2,2-Trifluoroethyl Nodulisporamide (7). Yield: 431 mg,
77%. TLC, silica gel: Rf ) 0.65 (5:95 MeOH/CHCl3). HPLC
(reversed-phase, ZORBAX Rx-C8, 4.6 mm × 25 cm, 6:4 to 1:0
MeCN/H2O, 20 min gradient, 2.0 mL/min, λ ) 254 nm) tR ) 11.4
1
°C). H NMR (500 MHz, CDCl3, ppm) δ 7.64 (s, 1H), 6.84 (d, J
) 10.7 Hz, 1H), 6.30 (dd, J ) 11.0, 15.3 Hz, 1H), 6.03 (d, J ) 2.9
Hz, 1H), 5.72 (d, J ) 15.5 Hz, 1H), 5.55 (s, 1H), 5.22 (dd, J )
2.1, 6.2 Hz, 1H), 5.18 (s, 1H), 5.06 (s, 1H), 4.97 (br s, 1H), 3.68
(s, 1H), 3.36 (br s, 1H), 3.16 (s, 1H), 2.86 (dd, J ) 2.7, 6.2 Hz,
1H), 2.82 (m, 1H), 2.70 (dd, J ) 6.5, 14.1 Hz, 1H), 2.28 (dd, J )
10.8, 13.7 Hz, 1H), 1.95-1.40 (m, 9H), 1.93 (s, 3H), 1.55 (s, 3H),

3514 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Meinke et al.
1
min. H NMR (500 MHz, CDCl3, ppm) δ 7.67 (s, 1H), 6.98 (d, J
5.06 (s, 1H), 5.00 (br s, 1H), 3.61 (m, 2H), 3.40 (m, 1H), 3.21 (s,
1H), 2.86 (dd, J ) 2.7, 6.1 Hz, 1H), 2.81 (m, 1H), 2.73 (dd, J )
6.3, 13.5 Hz, 1H), 2.40 (m, 1H), 2.28 (dd, J ) 10.8, 13.6 Hz, 1H),
1.99 (s, 3H), 1.95-1.40 (m, 11H), 1.57 (s, 3H), 1.47 (s, 3H), 1.34
(s, 3H), 1.32 (s, 3H), 1.13 (s, 3H), 1.12 (s, 3H), 1.05 (s, 3H), 0.93
(s, 3H). HRMS (ESI, pos mode) calculated for C46H57F3N2O5,
774.421 96; found m/z ) 775.429 73 [M + H]+.
) 11.2 Hz, 1H), 6.33 (dd, J ) 11.0, 15.3 Hz, 1H), 6.03 (d, J ) 3.0
Hz, 1H), 5.95 (t, J ) 6.3 Hz, 1H), 5.82 (d, J ) 15.4 Hz, 1H), 5.22
(d, J ) 6.0 Hz, 1H), 5.18 (s, 1H), 5.06 (s, 1H), 4.97 (br s, 1H),
4.00 (m, 2H), 3.38 (br s, 1H), 3.16 (s, 1H), 2.86 (dd, J ) 2.8, 6.2
Hz, 1H), 2.81 (m, 1H), 2.71 (dd, J ) 6.5, 13.8 Hz, 1H), 2.28 (dd,
J ) 11.0, 13.8 Hz, 1H), 1.99 (s, 3H), 1.95-1.40 (m, 10H), 1.56 (s,
3H), 1.47 (s, 3H), 1.34 (s, 3H), 1.32 (s, 3H), 1.12 (s, 3H), 1.11 (s,
3H), 1.04 (s, 3H), 0.93 (s, 3H). HRMS (ESI, pos mode) calculated
for C45H55F3N2O5, 760.4069; found m/z ) 761.41428 [M + H]+.
N-Isobutyl Nodulisporamide (8). Yield: 542 mg, 67%. TLC,
silica gel: Rf ) 0.24 (3:1 hexanes/acetone). HPLC (reversed-phase,
ZORBAX Rx-C8, 4.6 mm × 25 cm, 6:4 to 1:0 MeCN/H2O, 20
N-(1,1,2,2-Tetramethyl)hydroxyethyl Nodulisporamide (13).
Yield: 408 mg, 36%. TLC, silica gel: Rf ) 0.51 (1:1 acetone/
1
hexanes). H NMR (500 MHz, CDCl3, ppm) δ 7.72 (s, 1H), 6.99
(d, J ) 11.1 Hz, 1H), 6.36 (dd, J ) 11.0, 14.9 Hz, 1H), 6.05 (s,
1H), 5.82 (s, 1H), 5.80 (d, J ) 15.1 Hz, 1H), 5.65 (s, 1H), 5.27 (d,
J ) 6.1 Hz, 1H), 5.21 (s, 1H), 5.08 (s, 1H), 5.03 (br s, 1H), 3.41
(br s, 1H), 3.18 (m, 1H), 2.91 (d, J ) 3.5 Hz, 1H), 2.86 (m, 1H),
2.75 (dd, J ) 6.0, 13.6 Hz, 1H), 2.35 (dd, J ) 10.3, 14.1 Hz, 1H),
2.02 (s, 3H), 1.95-1.40 (m, 10H), 1.51 (s, 3H), 1.56 (s, 3H), 1.40
(s, 6H), 1.38 (s, 3H), 1.36 (s, 3H), 1.22 (s, 6H), 1.17 (s, 3H), 1.16
(s, 3H), 1.07 (s, 3H), 0.98 (s, 3H). HRMS (ESI, pos mode)
calculated for C49H66N2O6, 778.492 09; found m/z ) 779.499 18
[M + H]+.
1
min gradient, 2.0 mL/min, λ ) 254 nm) tR ) 22.6 min. H NMR
(500 MHz, CDCl3, ppm) δ 7.68 (s, 1H), 6.94 (d, J ) 11.7 Hz,
1H), 6.32 (dd, J ) 11.0, 15.1 Hz, 1H), 6.02 (d, J ) 2.8 Hz, 1H),
5.74 (m, 2H), 5.22 (dd, J ) 2.3, 6.3 Hz, 1H), 5.18 (s, 1H), 5.06 (s,
1H), 4.97 (br s, 1H), 3.68 (s, 1H), 3.39 (m, 1H), 3.16 (t, J ) 6.4
Hz, 2H), 2.86 (dd, J ) 2.7, 6.1 Hz, 1H), 2.81 (m, 1H), 2.70 (dd, J
) 6.5, 14.1 Hz, 1H), 2.28 (dd, J ) 10.7, 13.7 Hz, 1H), 1.97 (s,
3H), 1.95-1.40 (m, 11H), 1.54 (s, 3H), 1.47 (s, 3H), 1.34 (s, 3H),
1.32 (s, 3H), 1.13 (s, 3H), 1.11 (s, 3H), 1.03 (s, 3H), 0.93 (s, 3H),
0.92 (d, J ) 6.4 Hz, 6H). HRMS (ESI, pos mode) calculated for
C47H62N2O5, 734.4658; found m/z ) 735.473 10 [M + H]+.
N-(1-Fluoromethyl)-2-fluoroethyl Nodulisporamide (14).
Yield: 449 mg, 40%. TLC silica gel: Rf ) 0.29 (1:2 acetone/
1
hexanes). H NMR (500 MHz, CDCl3, ppm) δ 7.71 (s, 1H), 7.00
(d, J ) 11.0 Hz, 1H), 6.36 (dd, J ) 11.0, 15.4 Hz, 1H), 6.06 (s,
1H), 6.05 (d, J ) 10.1 Hz, 1H), 5.85 (d, J ) 15.3 Hz, 1H), 5.26
(d, J ) 6.2 Hz, 1H), 5.21 (s, 1H), 5.09 (s, 1H), 5.01 (br s, 1H),
4.48-4.71 (m, 5H), 3.41 (br s, 1H), 2.90 (dd, J ) 2.8, 6.0 Hz,
1H), 2.85 (m, 1H), 2.75 (dd, J ) 6.6, 13.9 Hz, 1H), 2.32 (dd, J )
11.0, 13.6 Hz, 1H), 2.01 (s, 3H), 1.95-1.40 (m, 11H), 1.62 (s,
3H), 1.51 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 1.16 (s, 3H), 1.15 (s,
3H), 1.08 (s, 3H), 0.97 (s, 3H). HRMS (ESI, pos mode) calculated
for C46H58F2N2O5, 756.431 38; found m/z ) 737.431 55 [M - H2O
+ H]+.
N-(1,1-Dimethyl)carbomethoxymethyl Nodulisporamide (9).
Yield: 17 mg, 42%. TLC, silica gel: Rf ) 0.65 (1:1 acetone/
hexanes). H NMR (500 MHz, CDCl3, ppm) δ 7.72 (s, 1H), 6.99
1
(d, J ) 10.8 Hz, 1H), 6.36 (dd, J ) 11.2, 15.1 Hz, 1H), 6.07 (s,
1H), 5.82 (d, J ) 15.3 Hz, 1H), 5.27 (d, J ) 5.9 Hz, 1H), 5.23 (s,
1H), 5.10 (s, 1H), 5.02 (br s, 1H), 3.93 (s, 3H), 3.73 (s, 1H), 3.41
(br s, 1H), 2.91 (d, J ) 3.7 Hz, 1H), 2.89 (m, 1H), 2.76 (dd, J )
6.1, 13.8 Hz, 1H), 2.34 (dd, J ) 10.7, 14.0 Hz, 1H), 2.01 (s, 3H),
1.95-1.40 (m, 11H), 1.60 (s, 6H), 1.57 (s, 3H), 1.51 (s, 3H), 1.38
(s, 3H), 1.36 (s, 3H), 1.16 (s, 3H), 1.15 (s, 3H), 1.07 (s, 3H), 0.97
(s, 3H). HRMS (ESI, pos mode) calculated for C48H62N2O7,
778.455 62; found m/z ) 779.462 90 [M + H]+.
Acknowledgment. We thank colleagues in Merck Research
Laboratories’ Natural Products group for their considerable
efforts dedicated toward the fermentation and isolation of
sufficient NsA A to perform the research described herein. We
gratefully acknowledge Dr. J. A. Hair, a leading expert in tick
biology (NuEra Laboratories, OK), for performing the flea and
tick study, whose results are described in Table 8. In addition,
we thank Deborah Zink for providing the high resolution mass
spectral analyses.
N-(1,1-Dimethyl)-2-oxopropyl Nodulisporamide (10). Yield:
412 mg, 41%. TLC, silica gel: Rf ) 0.52 (0.4:1 acetone/hexanes).
RP-HPLC (C18 ODS, 1 mL/min, 4:1 MeCN/H2O, isocratic): tR )
1
6.1 min. H NMR (500 MHz, CDCl3, ppm) δ 7.71 (s, 1H), 6.98
(d, J ) 10.8 Hz, 1H), 6.70 (s, 1H), 6.35 (dd, J ) 11.2, 15.1 Hz,
1H), 6.06 (s, 1H), 5.81 (d, J ) 15.3 Hz, 1H), 5.26 (d, J ) 5.9 Hz,
1H), 5.22 (s, 1H), 5.09 (s, 1H), 5.01 (br s, 1H), 3.72 (s, 1H), 3.40
(br s, 1H), 2.90 (d, J ) 3.7 Hz, 1H), 2.88 (m, 1H), 2.75 (dd, J )
6.1, 13.8 Hz, 1H), 2.33 (dd, J ) 10.7, 14.0 Hz, 1H), 2.23 (s, 3H),
2.01 (s, 3H), 1.95-1.40 (m, 10H), 1.60 (s, 6H), 1.57 (s, 3H), 1.51
(s, 3H), 1.38 (s, 3H), 1.36 (s, 3H), 1.16 (s, 3H), 1.15 (s, 3H), 1.07
(s, 3H), 0.97 (s, 3H). HRMS (ESI, pos mode) calculated for
C48H62N2O6, 762.460 79; found m/z ) 763.468 00 [M + H]+.
N-Cyclopropylmethyl Nodulisporamide (11). Yield: 87 mg,
47%. TLC, silica gel: Rf ) 0.49 (5:95 10% NH4OH in MeOH/
CHCl3). HPLC (reversed-phase, ZORBAX Rx-C8, 4.6 mm × 25
cm, 6:4 to 8:2 MeCN/H2O, 20 min gradient, 2.0 mL/min, λ ) 254
Supporting Information Available: Spectra of 3 (1H, 13C, DEPT
NMR spectra, UV-vis and IR spectra) (Figures III-VI); profiles
(flea and bedbug activity, T1/2 data, and flea efficacy in dogs) for
66 nodulisporamides (Tables I-IV); plasma disposition profiles for
nodulisporamides 3-14 in dogs (42 day study) and cats (28 day
study) (Tables V and VI); plasma disposition profile of 3 in dogs
(91 day multiple dosing study) (Table 7); tissue distribution of
[14C]3 in mice at 5 and 20 mg/kg (Figure I); plasma disposition
profile of 2 in dogs dosed at 30 mg/kg once or 15 mg/kg twice
(Table 8); plasma disposition profile of 3 dosed in dogs at 30 mg/
kg (90 day study) (Table IX and Figure II); plasma disposition
profile of 3 dosed in cats at 15 or 20 mg/kg (Table X); plasma
disposition profile of 3 dosed in dogs at 5 or 10 mg/kg (Table XI).
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
nm) tR ) 11.8 min. H NMR (500 MHz, CDCl3, ppm) δ 7.68 (s,
1H), 6.95 (d, J ) 11.2 Hz, 1H), 6.32 (dd, J ) 11.1, 15.5 Hz, 1H),
6.02 (d, J ) 3.0 Hz, 1H), 5.82 (t, J ) 5.5 Hz, 1H), 5.76 (d, J )
15.3 Hz, 1H), 5.23 (dd, J ) 2.1, 6.2 Hz, 1H), 5.18 (s, 1H), 5.06 (s,
1H), 4.97 (br s, 1H), 3.37 (br s, 1H), 3.17 (m, 2H), 2.86 (dd, J )
2.7, 6.2 Hz, 1H), 2.81 (m, 1H), 2.70 (dd, J ) 6.3, 14.1 Hz, 1H),
2.28 (dd, J ) 10.9, 13.7 Hz, 1H), 1.98 (s, 3H), 1.95-1.40 (11H,
m), 1.59 (s, 3H), 1.47 (s, 3H), 1.34 (s, 3H), 1.32 (s, 3H), 1.12 (s,
3H), 1.11 (s, 3H), 1.04 (s, 3H), 0.93 (s, 3H), 0.51 (q, J ) 8.0 Hz,
2H), 0.22 (q, J) 4.8 Hz, 2H). HRMS (ESI, pos mode) calculated
for C47H60N2O5, 732.450 22. found m/z ) 733.457 53 [M + H]+.
N-(3,3,3-Trifluoro)-n-propyl Nodulisporamide (12). Yield: 401
mg, 33%. TLC, silica gel: Rf ) 0.58 (6:4 hexanes/acetone). HPLC
(reversed-phase, ODS, 4.6 mm × 25 cm, 7:3 MeCN/H2O, isocratic,
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