Pilot in Vivo Structure-Activity Relationship of Dihydromethysticin in Blocking 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced O6-Methylguanine and Lung Tumor in A/J Mice

Article abstract of DOI:10.1021/acs.jmedchem.7b00921

(+)-Dihydromethysticin was recently identified as a promising lung cancer chemopreventive agent, while (+)-dihydrokavain was completely ineffective. A pilot in vivo structure-activity relationship (SAR) was explored, evaluating the efficacy of its analogs in blocking 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced short-term O⁶-methylguanine and long-term adenoma formation in the lung tissues in A/J mice. Both results revealed cohesive SARs, demonstrating that the methylenedioxy functional group in DHM is essential while the lactone functional group tolerates modifications.

Full text of DOI:10.1021/acs.jmedchem.7b00921

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Brief Article
Pilot in vivo structure-activity relationship of dihydromethys-
ticin in blocking 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone-induced O6-methylguanine and lung tumor in A/J mice
manohar puppala, Sreekanth C. Narayanapillai, Pablo Leitzman, Haifeng Sun,
Pramod Upadhyaya, M. Gerard O'Sullivan, Stephen S. Hecht, and Chengguo Xing
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00921 • Publication Date (Web): 14 Aug 2017
Downloaded from http://pubs.acs.org on August 14, 2017
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Journal of Medicinal Chemistry
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Pilot in vivo structure-activity relationship of dihydromethys-
ticin in blocking 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone-induced O6-methylguanine and lung tumor in A/J
mice
Manohar Puppala,1,# Sreekanth C. Narayanapillai,1,2,# Pablo Leitzman,1 Haifeng Sun,2 Pramod
Upadhyaya,3 M. Gerard OˈSullivan,4,5 Stephen S. Hecht,3 and Chengguo Xing,1,2*
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1Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455
2Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610
3Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
4Masonic Cancer Center Comparative Pathology Shared Resource, University of Minnesota, St. Paul, MN 55108
5Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN 55108
ABSTRACT: (+)-Dihydromethysticin was recently identified as a promising lung cancer chemopreventive agent while (+)-
dihydrokavain was completely ineffective. A pilot in vivo structure-activity relationship (SAR) was explored, evaluating
the efficacy of its analogs in blocking 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced short-term O6-
methylguanine and long-term adenoma formation in the lung tissues in A/J mice. Both results revealed cohesive SARs,
demonstrating that the methylenedioxy functional group in DHM is essential while the lactone functional group tolerates
modifications.
mechanism to initiate lung tumorigenesis.4 Inhibiting the
ꢀ
INTRODUCTION
formation of such DNA damage is, therefore, a plausible
strategy to reduce lung cancer risk. Along the journey of
understanding NNK carcinogenesis, Hecht and his col-
leagues have developed robust methods to quantify dif-
ferent DNA modifications via liquid chromatography-
electrospray ionization/tandem mass spectrometry (LC-
ESI-MS/MS) analyses of the hydrolytic products from
damaged DNA.
Lung cancer causes ~160,000 deaths in the U.S. each
year.1,2 Moreover, its five-year survival has been hovering
around 15% for decades.2 Given the limited success in ear-
ly diagnosis and treatment, prevention is of paramount
importance. Tobacco cessation is the best strategy to re-
duce lung cancer risk.3 However, many smokers will not
succeed in quitting because of the addictive nature of
nicotine in tobacco products. Chemopreventive agents,
therefore, are needed for these high-risk populations.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1
in Scheme 1) is a tobacco-specific carcinogen that induces
primarily lung tumor formation in various species.4 It has
been proposed to contribute to lung adenocarcinoma
incidence among smokers in the U.S.A.5 Mechanistically
NNK needs metabolic activation via cytochrome P450
(CYP450) enzyme-mediated hydroxylation to generate
two reactive species, which can react with DNA leading to
DNA damages, including methylation and pyridyloxobu-
tylation (POB, Scheme 1).3,6 NNK can also be reduced to
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 2),
which is likely the dominant metabolic pathway of NNK
in humans.7 Upon CYP450-mediated hydroxylation,
NNAL generates two reactive species as well, resulting in
DNA modifications, including methylation and pyri-
dylhydroxybutylation (PHB). DNA damage induced by
NNK and NNAL has been proposed as the underlying
Scheme 1. Simplified metabolism of NNK and NNAL, focus-
ing on the bioactivation, generation of reactive intermedi-
ates, and subsequent formation of different types of DNA
damage.
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Among lung tumorigenesis models, the NNK-induced A/J adduct of 8 was detected via LC-MS/MS when (+)-DHM
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mouse model is widely used in identifying lung cancer
chemopreventive agents while the tobacco smoke-
induced model is less practical.8-10 Because of the predis-
posed pulmonary adenoma susceptibility 1 (Pas1) gene,9
the A/J mice are highly susceptible to lung tumorigenesis.
They can develop lung adenomas in a relatively short time
with 100% incidence and high adenoma multiplicity upon
appropriate NNK treatment.8,10 Although the contribution
of each type of DNA damage to lung tumorigenesis has
not been quantified, there has been compelling evidence
suggesting that O6-mG is the most carcinogenic in A/J
mice.11-16
was incubated in human liver microsomes in the presence
of glutathione.25 Such a result suggest that the meth-
ylenedioxy functional group in (+)-DHM and (+)-
methysticin are likely subjected to metabolism in vivo. In
addition, molecules of the same molecular weights as in-
termediates 9 and 10 have been detected via LC-MS/MS
by the Xing group when (+)-DHM was incubated in
mouse liver microsomes (data not shown), suggesting
that the lactone group in (+)-DHM is metabolically labile
as well. It therefore remains to be determined whether
intact (+)-DHM or any of its potential metabolites (7 – 10)
is the active form responsible for its chemopreventive
potential.
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Figure 1. Structures of four major natural kavalactones in
kava.
We have recently identified (+)-dihydromethysticin ((+)-
DHM, 3 in Figure 1) from kava as a promising lung cancer
chemopreventive agent. (+)-DHM potently and selectively
inhibited NNK-induced O6-mG formation in the target
lung tissues.17 (+)-DHM also completely blocked lung ad-
enoma formation in A/J female mice at a dose of 0.05
mg/g of diet.17 Interestingly, several structurally similar
lactone compounds in kava revealed a clear structure ac-
tivity relationship (SAR).17 (+)-Dihydrokavain ((+)-DHK,
4), for instance, was completely ineffective in inhibiting
O6-mG and lung adenoma formation even at the dose of
0.5 – 1.0 mg/g of diet.17 (+)-Methysticin (5) was able to
significantly inhibit NNK-induced O6-mG formation, only
slightly less effective than (+)-DHM, while (+)-kavain (6)
was not effective at all. These results overall suggest that
the methylenedioxy functional group in (+)-DHM and
(+)-methysticin is critical for their lung cancer chemopre-
ventive potential.
Scheme 2. Proposed metabolism of DHM and the corre-
sponding metabolites.
These questions led to our current effort to investigate
the role of the methylenedioxy and the lactone of DHM
on its lung cancer chemopreventive activity. We have
synthesized (±)-DHM (11), (±)-DHK (12) and three ana-
logs (13-15) (Figure 2). By characterizing their effect on
NNK-induced O6-mG and adenoma formation in the tar-
get lung tissue in A/J mice, a cohesive in vivo SAR of DHM
in preventing lung tumorigenesis was observed, which
provides key structural insights that will facilitate future
mechanistic investigations and structural optimizations.
ꢀ
RESULTS AND DISCUSSION
Rational design Since any of the metabolites or intact
DHM in Scheme 2 could be the in vivo active form, we
proposed three analogs to probe these possibilities (Fig-
ure 2A, 13-15). Compound 13 would block methylene hy-
droxylation, compound 14 would block lactone hydrolysis,
while compound 15 would block both pathways of metab-
olism.
Kava traditionally is an aqueous suspension of the root of
“noble” cultivars of piper methysticum and has been con-
sumed as a beverage in the South Pacific Islands to help
people relax and improve the quality of sleep.18 The or-
ganic (ethanol or acetone) extract of the root had been
used as an anxiolytic agent in Europe with some debata-
ble hepatotoxic risk.19,20 The organic extract form is cur-
rently available in the US as a dietary supplement.21 Alt-
hough there has been a long history of kava usage in hu-
mans with a number of clinical trials,22 systematic charac-
terization of its pharmacokinetics and metabolism has
been limited, particularly with respect to (+)-DHM and
(+)-methysticin because they are not believed to be re-
sponsible for kava’s relaxing and anxiolytic properties.23
Nevertheless, the methylenedioxy functional group can be
metabolized by CYP enzymes via methylene hydroxyla-
tion followed by ring opening to generate a hydroquinone
intermediate. In the case of DHM, intermediate 7 would
be generated, which can be oxidized to 8 (Scheme 2). Alt-
hough intermediates 7 and 8 have not been detected in
humans upon kava exposure,24 a glutathione-conjugated
Figure 2. Structures of racemic DHM, racemic DHK and
three racemic analogs (A) and their effect on NNK-induced
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O6-mG in the lung tissues in A/J mice (B). Quantification of
13 started with indane aldehyde in a 32% overall yield,
O6-mG in the target lung tissues in A/J mice (n=3 for 11 – 15
at a dose of 200 ppm and n=6 for NNK control group, O6-mG
was not detectable in the negative control group, data not
shown). Statistical analysis was performed with ONE-WAY
ANOVA followed by Dunnett’s test of each treatment group
relative to the NNK control group; *** p<0.001; **** p<0.0001.
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following the same synthetic route in Scheme 3B. Analogs
14 and 15 were synthesized via a different route (Scheme
3C). Using 14 as the example, aldehyde 19 upon ylide
treatment via the Wittig reaction generated olefin ester 21
in a 95% yield. Intermediate 21 was converted to cyclic
diketone 22 upon a sequential Michael addition and
Dieckmann condensation, which was then methylated to
furnish 14 in a 30% overall yield.
9
Short-term effect of 11-15 on NNK-induced O6-mG in
the target lung tissues Given the carcinogenic im-
portance of O6-mG in NNK-induced lung tumorigenesis
in A/J mice and the distinct effects between (+)-DHM and
(+)-DHK on O6-mG,17 11-15 were first evaluated for their
effect on NNK-induced O6-mG in the target lung tissues
in A/J mice at the dose of 200 ppm in diet, following our
reported procedures (Figure 2B).17 Consistent with our
previous results for (+)-DHM and (+)-DHK, 11 effectively
reduced O6-mG adduct formation (72.3% reduction, p <
0.0001) while 12 treatment resulted in a minimal and non-
significant reduction (16.4% reduction, p > 0.05). Com-
pounds 13 and 15 also induced minimal and non-
significant reductions in O6-mG adducts (29.6% and 11.8%
reduction respectively, p > 0.05). Compound 14, on the
other hand, resulted in a significant reduction in O6-mG
adduct formation (63.8%, p < 0.001) relative to the NNK
control group and statistically not different from 11.
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Scheme 3. The synthesis of 11 – 15. (A) The first synthetic
route for 11 with 16 being a byproduct: (i) (a) (1,3-dioxolan-2-
yl)methyl triphenylphosphonium bromide, LiOMe, 70 °C 6h;
(b) 1N HCl, THF, RT, 2h; (c) Ethyl acetoacetate, NaH, n-BuLi,
THF -78°C to RT; (d) K2CO3/methanol, then dimethyl-
sulfate/acetone; (ii) H2, Pd/C, THF. (B) The second synthetic
route of 11: (i) LiOMe, dry THF, 0 °C, piperonal, 70 °C, 6 h;
(ii) H2, Pd/C (1 atm), THF, 2 h; (iii) 1N HCl, THF, RT, 2 h; (iv)
(a) NaH, n-BuLi, dry THF, 0 °C, 30 min, (b) -55 °C to RT, 6h;
(v) (a) K2CO3, MeOH, RT, 6h, (b) Dimethyl sulfate, acetone,
RT, 12 h. (C) The synthetic route of 14 and 15: (i) (carbethox-
ymethylene)triphenylphosphorate, THF, reflux, 12h; (ii) NaH,
acetone, THF and toluene, 0 °C-RT, 4h; (iii) Dimethyl sulfate,
acetone, RT, 12 h.
Concise syntheses We have previously developed a
heavy-metal free synthesis to obtain (±)-methysticin
(Scheme 3),26 which has been applied to the synthesis of
(±)-kavain. We initially envisioned that 11 and 12 could be
readily obtained from (±)-methysticin and (±)-kavain via
a selective reduction of the 7,8-alkenyl functional group
by catalytic hydrogenation (Scheme 3A). 12 indeed was
efficiently prepared from (±)-kavain as such. Hydrogena-
tion reaction, however, did not work well for the synthe-
sis of 11 even upon several attempts of optimization; a
considerable amount of an undesired hydrogenolytic
product, 16, was formed. The formation of 16 not only
decreased the yield of 11 but also complicated its purifica-
tion. The mechanism behind the different hydrogenation
outcomes of (±)-methysticin and (±)-kavain requires fur-
ther investigation. We changed the reaction sequence for
the synthesis of 11 to avoid the formation of 16 (Scheme
3B). The Wittig olefination of piperonal with the phos-
phorous ylide, followed by a quick column filtration, gave
the olefinic acetal 17. Compound 17 upon catalytic hydro-
genation furnished saturated acetal 18, which upon
deprotection with 1 N HCl treatment to afford the satu-
rated aldehyde 19 in a three-step 50% overall yield. Com-
pound 19, without further purification, was subjected to
dianion alkylation with ethyl acetoacetate, resulting in
the aldol product 20. K2CO3 mediated lactonization of 20
followed by methylation using dimethyl sulfate in acetone
afforded 11 in a 64% yield from 19. The synthesis of analog
Figure 3. Preliminary safety data of 11 – 14 (A – C) and their
impact on tumor multiplicity (D) (n=5 for 11 – 14 and n=10 for
NNK group). Statistical analysis was performed with ONE-
WAY ANOVA, followed by Dunnett’s test of each treatment
group relative to the NNK control group; **** p<0.0001.
Safety of 11 – 14 and their effect on the long-term lung
adenoma formation induced by NNK Based on their
short-term effect on O6-mG, 15 was not evaluated for its
effect on NNK-induced long-term lung adenoma for-
mation in A/J mice because of its least reduction in O6-
mG. 11 – 14 were evaluated at a dose of 25 ppm in diet
herein. Such a dose would be equivalent to a dose of ~ 20-
30 mg/day for a human of 75 kg bodyweight according to
the Body Surface Area Normalization method.27 Based on
their impact on the bodyweight and liver weight, all four
compounds were well tolerated (Figure 3A-C). Consistent
with the short-term O6-mG results and our previous
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study,17 11 effectively blocked NNK-induced long-term
brated using an internal reference. Chemical shifts (δ val-
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adenoma formation (94.2% reduction, p < 0.0001) while 12
was completely ineffective (6.3% reduction, p > 0.05).
Compound 13 also had minimal effect on adenoma multi-
plicity (10.1% reduction, p > 0.05) while 14 significantly
reduced adenoma multiplicity (89.4% reduction, p <
0.0001).
ues) and coupling constants (J values) were given in ppm
and hertz, respectively. ESI mode mass spectra were rec-
orded on a Bruker BiotofII mass spectrometer. All com-
pounds synthesized are racemic mixtures and are more
than 95% pure, analyzed using HPLC. Detailed proce-
dures and characterizations see Supporting Information.
7
8
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A/J mouse in vivo studies The animal studies performed
herein were approved by the University of Minnesota In-
stitutional Animal Care and Use Committee and conduct-
ed following the National Institutes of Health guidelines.
AIN-G and AIN-M powdered diet was purchased from
Harlan Teklad (Madison, WI). Female A/J mice at the age
of 6 weeks were purchased from Jackson Laboratory (Bar
Harbor, Maine).
ꢀ
CONCLUSION
In this study, we investigated the importance of the
methylenedioxy and lactone functional groups on the
chemopreventive activity of DHM using three rationally
designed synthetic analogs that can respectively block
either methylene hydroxylation, lactone hydrolysis or
both routes of metabolism. To achieve this goal, we first
developed facile syntheses of these analogs. Their lung
cancer chemopreventive properties in comparison to the
racemic DHM (active) and the racemic DHK (inactive)
were then evaluated under the short-term and long-term
conditions using a well-established NNK-induced lung
tumorigenesis A/J mouse model. Compounds 13 and 15,
devoid of the dioxy functional group of DHM while re-
taining the five-membered ring, did not show any signifi-
cant inhibitory activity against NNK-induced O6-mG for-
mation or lung tumor multiplicity in A/J mice. Interest-
ingly compound 14, with the intact methylenedioxy func-
tional group but the mask of the lactone functional group,
recapitulated the O6-mG adduct reduction potential and
antitumorigenic efficiency of DHM. It is noteworthy to
restate that DHK, which lacks the methylenedioxy group,
is inactive against NNK-induced DNA adducts and tu-
morigenesis. From these results, it is clear that the meth-
ylenedioxy functional group of DHM is critical for its
chemopreventive activity while the lactone functional
group tolerates modifications. In addition, the reduction
in O6-mG correlates well with the blockage of lung ade-
noma formation, consistent with the results from Peter-
son et al. that O6-mG reduction could serve as a short-
term surrogate to screen for effective chemopreventive
candidates.11 Together, the results of this study reveal a
cohesive structure-activity relationship of DHM for its
chemopreventive potential. Studies are ongoing to de-
termine whether the intact methylenedioxy moiety of
DHM is involved in the direct interactions with the cellu-
lar target or the methylenedioxy functional group in
DHM is metabolized to reveal the in vivo active form.
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Diet preparation and characterization AIN-G pow-
dered diets supplemented with 11 – 15 respectively were
prepared following our reported procedures.8 Briefly, each
compound was reconstituted in absolute ethanol (50 mL)
and then mixed with the AIN-93 G powdered diet (150 g).
Absolute ethanol (50 mL) was mixed with the AIN-93 G
powdered diet (150g) for the control diet. The reconstitut-
ed diets were dried under vacuum to remove ethanol and
then ground into fine powders. All diets were then mixed
well with additional AIN-93 G powdered diet to the de-
sired dose. The abundance of the respective compound in
the diet was analyzed in triplicate by HPLC and con-
firmed to be within ± 10% of the specified dose.
Caution: NNK is IACR Group 1 carcinogen. Personnel
handlings are expected to have appropriate safety
measures. NNK and [CD3]O6-mG were purchased from
Toronto Research Chemicals (Toronto, ON, Canada).
Impact of 11 – 15 on NNK-induced O6-mG DNA adduct
in the lung tissues After one week of acclimation, 27
female A/J mice (6-7 weeks of age) were randomized into
seven groups each (n=3 except for the negative and NNK
control groups n=6) and maintained on the specific diet
starting on Day 0 (control diet for negative and NNK con-
trol groups, or the corresponding diet supplemented with
11 – 15 respectively at a dose of 200 ppm). Bodyweight of
the mice was measured every two or three days and their
food intake was monitored twice a week. On Day 7, ex-
cept for mice in the negative control groups, all mice re-
ceived a single dose of NNK in saline (100 µL) at the dose
of 100 mg/kg of bodyweight via i.p. injection. Mice in the
negative control group were given saline. Based on the
results from our previous studies,28 four hours after NNK
exposure, mice were euthanized with CO2 overdosing.
The lung tissues were collected and stored at -80 °C till
analysis. DNA isolation from the lung tissues and LC-
MS/MS quantification were performed following the
standard procedure.17 Briefly, DNA was isolated from half
of the whole lung tissue of each individual mouse, follow-
ing the Puregene DNA isolation protocol (Qiagen Corp).
O6-mG was quantified by LC-ESI-MS/MS with [CD3]O6-
mG as the internal standard.
ꢀ
EXPERIMENTAL SECTION
Chemistry All commercial reagents and anhydrous sol-
vents were purchased from vendors and were used with-
out further purification or distillation unless otherwise
stated. Analytical thin layer chromatography was per-
formed on Whatman silica gel 60 Å with fluorescent indi-
cator (partisil K6F). Compounds were visualized by UV
light and/or stained with potassium permanganate solu-
tion followed by heating. Flash column chromatography
was performed on Whatman silica gel 60 Å (230-400
mesh). NMR (1H and 13C) spectra were recorded on a Vari-
an 400 MHz or a Bruker 500 MHz spectrometer and cali-
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Synthesis, characterization and molecular formula strings are
Impact of 11 – 14 on NNK-induced adenoma for-
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3
4
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6
7
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included, available free of charge via the Internet at
http://pubs.acs.org.
mation in the lung tissues and preliminary safety
monitoring After one week of acclimation, 35 female A/J
mice (6-7 weeks of age) were randomized into six groups
each (n=5 each group except for the NNK-alone group,
n=10) and maintained on the specific diet at a dose of 25
ppm, starting on Day 0. NNK was given at a dose of 100
and 67 mg/kg bodyweight via i.p. injection on Day 7 and
Day 14 respectively. The dietary treatment stopped 24
hours after the last NNK treatment and mice were main-
tained on standard AIN-M powdered diet. Food intake
was monitored twice a week and bodyweight was moni-
tored once a week. At the end of Day 119, mice were
weighed and euthanized via CO2 overdosing. The liver
tissues were collected and weighed. The relative liver
weight was calculated as the ratio of the liver weight and
bodyweight of each mouse. Adenomas on the surface of
the lung were counted under blinded conditions by an
A.C.V.P board certified pathologist (M.G. O’S.).
ꢀ
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Statistical analysis Data on the quantity of O6-mG and
lung adenoma multiplicity were reported as mean ± SD.
One-way ANOVA was used to compare means among
NNK and NNK + treatment groups. The Dunnett’s test
was used for comparisons of the quantity between NNK
control and NNK + treatment groups. P value ≤0.05 was
considered statistically significant. All analyses were con-
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
Corresponding Authors
Phone, 352-294-8511; E-mail, chengguoxing@cop.ufl.edu.
ORCID
Chengguo Xing: 0000-0002-4266-6236
Notes
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
(12) Liu, L.; Qin, X.; Gerson, S. L. Reduced lung tumorigenesis
in human methylguanine DNA--methyltransferase transgenic
mice achieved by expression of transgene within the target cell.
Carcinogenesis 1999, 20, 279-284.
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ACKNOWLEDGMENT
(13) Loechler, E. L.; Green, C. L.; Essigmann, J. M. In vivo muta-
genesis by O6-methylguanine built into a unique site in a viral
genome. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 6271-6275.
This work was funded by grants from National Institutes of
Health Grant R01-CA193278 (Xing) and in part by the Na-
tional Cancer Institute Cancer Center Support Grant CA
077598 (Turesky). We thank Dr. Peter Villalta of the Masonic
Cancer Center’s Analytical Biochemistry shared resource for
direction on the use of LC-MS/MS instrument.
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(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related
DNA methylating and pyridyloxobutylating agents. Carcinogene-
sis 1993, 14, 2419-2422.
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ABBREVIATIONS
SAR, structure-activity relationship; DHM, dihydromethys-
ticin; DHK, dihydrokavain; NNK, 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone; NNAL, 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanol; O6-mG, O6-methylguanine; LC-MS/MS,
liquid chromatography–tandem mass spectrometry; ANOVA,
analysis of variance.
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son, L. A.; Lu, J.; Hecht, S. S.; Xing, C. Dihydromethysticin from
kava blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone-induced lung tumorigenesis and differen-
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Supporting Information Availability
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