Potent and selective inhibition of human cytochrome P450 3A4 by seco-pancratistatin structural analogs

Article abstract of DOI:10.1016/j.bmcl.2010.01.157

seco-Derivatives of the anticancer agent pancratistatin bearing the 2S,3S,4S,5S configuration were accessed via a novel, highly diastereoselective anti-aldol reaction. Structure-activity relationships reveal important insights into the seco-pancratistatin pharmacophore as a potent and selective inhibitor of human cytochrome P450 3A4 (CYP3A4), and highlight features of concern in advancing a potent, selective anticancer agent in the pancratistatin series.

Full text of DOI:10.1016/j.bmcl.2010.01.157

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2335–2339
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and selective inhibition of human cytochrome P450 3A4
by seco-pancratistatin structural analogs
a
b
b
b
James McNulty a, , Jerald J. Nair , Mohini Singh , Denis J. Crankshaw , Alison C. Holloway
*
a Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ont., Canada L8S 4M1
b Department of Obstetrics and Gynecology, McMaster University, 1200 Main Street West, Hamilton, Ont., Canada L8N 3Z5
a r t i c l e i n f o
a b s t r a c t
Article history:
seco-Derivatives of the anticancer agent pancratistatin bearing the 2S,3S,4S,5S configuration were
accessed via a novel, highly diastereoselective anti-aldol reaction. Structure–activity relationships reveal
important insights into the seco-pancratistatin pharmacophore as a potent and selective inhibitor of
human cytochrome P450 3A4 (CYP3A4), and highlight features of concern in advancing a potent, selective
anticancer agent in the pancratistatin series.
Received 17 December 2009
Revised 21 January 2010
Accepted 27 January 2010
Available online 4 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pancratistatin
Human cytochrome
P450 3A4
Anticancer agent
Antioxidant
Antifungal
The plant family Amaryllidaceae produces an array of structur-
ally-diverse alkaloids arising biosynthetically from the common
amino acid-derived precursor norbelladine 1 (Scheme 1).1 Galan-
thamine 2, crinine 3, and lycorine 5 are representative of the three
major structural types within the family although six other minor
groups are known to occur, including homolycorine 8, tazettine 9,
and montanine 10.1 The interesting biological properties exhibited
by several alkaloids of the Amaryllidaceae has garnered wide inter-
est in terms of the phytochemical, synthetic, semi-synthetic, and
biological evaluation aspects of these compounds, areas in which
we have been active for a number of years now.2 In particular,
the cell-line specific anticancer activity displayed by pancratistatin
6 and narciclasine 7, as well as their challenging molecular struc-
ture has attracted sustained synthetic efforts over the past two
decades.3 Galanthamine 2 is the first representative of this group
of alkaloids to receive approval as a scheduled drug in the treat-
ment of Alzheimer’s disease due to its ability to reversibly inhibit
acetylcholinesterase.1c Amongst the other series within the Ama-
ryllidaceae, crinane compounds (such as 3) are known to inhibit
protein synthesis and have antimalarial and antiproliferative activ-
ity.4 Recent findings in our laboratories uncovered the selective
Lycorine 5 is the most common alkaloid across the Amaryllidaceae
and its broad spectrum of biological activities is well docu-
mented.4d,e,5a–l It has been shown to be a promising chemothera-
peutic agent with antiproliferative action in a number of cancer
cell lines.4e Lycorine has recently been identified as a selective5k
initiator of apoptosis in human leukemia (Jurkat) cells and as a
selective cytostatic agent in a mini-panel of cancer cell lines.5l
The essential elements of this remarkable pharmacophore have
been documented in two recent reports.5k,l
Following on the commercial success of galanthamine 2 and gi-
ven the potent and selective anticancer activity of pancratistatin 6
and narciclasine 7, it is widely held that an anticancer clinical can-
didate will emerge from the lycorane group. Pharmacophore min-
imization efforts by us and others have revealed several crucial
elements of this promising anticancer pharmacophore; in particu-
lar, the stereodefined 2,3,4-trihydroxy functionalized ring C is
essential for potent apoptosis-inducing activity.2a–e This was
shown by us to occur selectively in cancer cells but not in normal
cells and that the process was localized within mitochondria of
cancerous cells.6 Early activation of caspase-3 followed by flipping
of phosphatidyl serine to the outer leaflet of the plasma membrane
were diagnostic of the apoptotic mode of death initiated by panc-
ratistatin.6 It is also important to note recent studies that show
that narciclasine disrupts organization of the actin skeleton in can-
cer cells at lower (30–90 nM) concentrations.3s Narciclasine has
also been shown to increase survival in preclinical models of
human glioblastoma multiforme and clearly has much potential.
apoptosis-initiating ability of
a-bridged crinanes (such as 4) in
rat liver hepatoma (5123tc) but not in normal HEK293t cells, nota-
bly distinguishing them from inactive b-variants (such as 3).4f,g
* Corresponding author. Tel.: +1 905 525 9140Â27393; fax: +1 905 522 2509.
E-mail address: jmcnult@mcmaster.ca (J. McNulty).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.157

2336
J. McNulty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2335–2339
OH
OH
OCH3
OH
HO
11
O
N
CH3
O
O
O
O
HO
HO
H
H
12
N
N
N
H
H3CO
2. Galanthamine
1. Norbelladine
3. Crinine
4. Crinamine
OH
HO
OH
OH
H3C
N
H
1
3
OH
HO
OH
OH
2
10b
10
C
4
9
8
O
O
O
O
H3CO
H3CO
OH
4a
11
10a
A
B
N
NH
6
NH
O
O
O
6a
7
12
OH
O
OH
O
O
5. Lycorine
6. Pancratistatin
7. Narciclasine
8. Homolycorine
OCH3
OCH3
OH
H
N
CH3
O
O
H3CO
H3CO
OH
O
H
N
9. Tazettine
10. Montanine
Scheme 1. Structurally-diverse alkaloid representatives of the plant family Amaryllidaceae.
In view of the potential clinical development of an anticancer
agent within the lycorane series,3a–c we recently documented for
the first time the human cytochrome (CYP3A4) inhibitory activity
of a compound library consisting of 26 amaryllidaceae alkaloids
and derivatives.8 Cytochromes P450 (CYP450) are heme-contain-
ing mono-oxygenase enzymes that are expressed primarily on
smooth ER membranes of hepatocytes and by cells along the intes-
tinal tract mucosal surface.7a They are involved in the detoxifica-
tent with our earlier observations with lycorine derivatives.8 None-
theless, we highlight that the two series differ with respect to the
absolute configuration at both C2 and C3.
Herein we report an entry to the C2/C3 diastereomeric series of
seco-pancratistatin analogs using a non-auxiliary directed ap-
proach and identify MOM benzamide 32 as a potent CYP3A4 inhib-
itor (Ki 0.03 lM), and overall provide a more comprehensive view
of this important antioxidant pharmacophore. Esterification of
(3,4-methylenedioxy)phenylacetic acid 11 with 2-bromopropane
under basic conditions in acetonitrile gave isopropyl (3,4-methyl-
enedioxy)phenylacetate 26 in 95% isolated yield (Scheme 3). The
tion of
a wide variety of xenobiotics such as drugs and
environmental toxins.7b The CYP3A subfamily comprises about
30% of the total liver cytochrome P450 enzyme pool in humans,
and the isoenzyme CYP3A4 accounts for approximately 60% of
drugs metabolized.7b In addition, an estimated 70% of CYP protein
in the small intestinal epithelium is formed by this isoenzyme.7c
Thus, evaluation of CYP3A4-drug interactions is critical to the iden-
tification of a selective clinical candidate. From our initial study8
we documented that pancratistatin 6 exhibited low interaction
with CYP3A4 while narciclasine 7 proved to be a potent inhibitor.
In addition, we determined that lipophilic substitution at positions
1 and 2 in the lycorine series 5 resulted in the formation of potent
CYP3A4 inhibitors one magnitude less active than the clinically
used antifungal P450 inhibitor ketoconazole.8 We also examined
a mini-panel of conformationally flexible semi-synthetic seco-
pancratistatin derivatives uncovering further structural informa-
tion concerning CYP3A4 inhibitory activity.9 The seco-analogs were
prepared via a non-Evans’ anti-selective aldol10 reaction that gave
anti-adduct 16 as the major diastereomer (95:5) in 95% isolated
yield (Scheme 2, step c).2f,g Elaboration of adduct 16 (Scheme 2)
led to the seco-pancratistatin target 25 which possesses all of the
essential elements of the known anticancer pharmacophore,
including the stereodefined 1,2,3,4-tetrahydroxy motif and the
amide functionality, but in a conformationally mobile form.2g,9 It
was shown that seco-pancratistatin 25 exhibited no CYP3A4 inhib-
itory activity. In contrast, the seco-analogs 18 and 20 were found to
reaction of the lithium enolate of 26 with L-threose aldehyde 15
was found highly diastereoselective leading to the anti-adduct 27
as a single diastereomer in 90% yield (Scheme 3, step b).11 Only a
few reports exist involving the use of phenylacetic esters in aldol
chemistry. This may be due to the modest diastereoselectivities
observed through formation of mixtures of E- and Z-enolates.12
Previous reports on nucleophilic addition to aldehyde 15 have ex-
plained observed selectivities through intrinsic diastereoselectivity
in accord with the Felkin–Ahn model.13 To the best of our knowl-
edge, this work is the first document of an aldol between a phen-
ylacetic ester and L-threose aldehyde 15. Protection of the 3-
hydroxy group in 27 as the MOM ether (Scheme 3, step c) followed
by LiAlH4 reduction of ester 28 gave alcohol 29 in 93% yield. The
primary hydroxyl group in 29 was converted to the bromide 30
via nucleophilic bromination with NBS/triphenylphosphine/TEA,
and underwent smooth displacement with sodium azide over 5 h
at room temperature to give in 93% isolated yield azide 31. Reduc-
tion of azide 31 over 10% palladium on carbon and subsequent
benzoylation of the free amine secured MOM-benzamide 32 in
near quantitative yield. In a similar fashion, carbamate 34 was
obtained from azide 31 via the free amine by reaction with
Boc-anhydride and TEA. Double recrystallization of 34 from ethyl
acetate provided
a sample for single crystal X-ray analysis
be as potent as ketoconazole (Ki 0.03 and 0.07
lM, respectively) at
(Fig. 1) confirming it to be the 2S,3S stereoisomer, and providing
unequivocal proof of the initial anti aldol. Desilylation of MOM-
benzamide 32 with TBAF proceeded smoothly to the hydroxyben-
zamide 35 which was then functionalized as the O-tosylate 36.
Straightforward global deprotection of 32 with 2 M HCl led to
the inhibition of CYP3A4.9 This result justifies the utility of these
seco-amaryllidaceae analogs as probes useful in highlighting
CYP3A4 interactions. This study revealed lipophilic substitution
at C3 as a key factor for potent CYP3A4 inhibition,9 results consis-

J. McNulty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2335–2339
2337
O
N
H
O
N
O
OH
O
13
O
O
O
O
O
O
O
a
O
O
14
O
12
O
b
11
O
O
15
O
TMSO
O
OTBS
OTBS
O
O
O
O
O
c
N
O
16
OH
4
3
HO
OH
O
O
O
O
O
O
5
1
2'
2
6
MOMO
HO
MOMO
3'
O
O
OR
OR
OTBS
OH
O
1'
O
O
O
HN
O
O
6'
4'
5'
1"
HN
HN
HN
O
O
6"
5"
2"
3"
25
O
O
O
24
4"
17. R=H
22. R=H
23. R=TBS
18. R=TBS
19. R=TBDPS
20. R=Tos
21. R=Ac
Scheme 2. Synthesis of seco-pancratistatin analogs via Evans’ catalytic anti-aldol reaction as key step. Reagents and conditions: (a) PivCl, TEA, DEE, À78 °C to rt, 2 h; (b)
LiHMDS, THF, À78 °C to rt, 6 h, 84%; and (c) MgCl2, TEA, TMSCl, EtOAc, rt, 12 h, 95%.
the tetrahydroxybenzamide 33 (89%), yielding the targeted C2/C3
diastereomeric variant of seco-pancratistatin 25 and analogs
shown.
The mini-library comprising functionalized truncated com-
pounds 27–36 (Scheme 3) was screened for CYP3A4 activity as pre-
viously reported (see Supplementary data), utilizing ketoconazole
as control (Table 1).8,9 The inhibitory activity of MOM-benzamide
small, polar group such as acetate 21 results in further loss of activ-
ity (Ki 1.01 M). While the OTBS-substituent at C6 appears optimal,
inhibition is maintained with the introduction of an O-tosylate
group at C6 as seen in both 36 (Ki 0.07 M) and 20.
l
l
Cleavage of both 3-MOM and 6-TBS groups resulted in complete
lack of activity as shown for 3,6-diol 22 (Scheme 2) while a further
loss of the 3,4-isopropylidene group also produced inactive tetra-
hydroxybenzamide 25.9 These results are in accord with our previ-
ous findings wherein a small H-bond acceptor at C3 was found
optimal. The fact that 25 and 33 were both inactive again high-
lights the requirement for lipophilic substitution around C3/C4/
C5 in the seco-pancratistatin series. The isopropylidene group
may also serve as a conformational anchor within a more rigid por-
tion of the enzyme.
32 (Ki 0.03 lM) is immediately striking, seen in Table 1 to be as po-
tent as ketoconazole. Compounds 27–31 were all inactive as was
seco-pancratistatin 33. A pronounced ꢀ60-fold reduction in activ-
ity is observed in going from MOM-benzamide 32 to desilylated
hydroxy analog 35 (Ki 1.78
introduction of an O-tosylate group at C6 as in 36 (Ki 0.07
l
M). Inhibition is sustained with the
M)
l
while replacement of the benzamide moiety in 32 with a t-butyl
carbamate resulted in a ꢀ10-fold decrease in activity as evident
While all of the above data show a consistent pattern of CYP3A4
activity, perhaps the most striking result is the total lack of relation
of this activity to either the 2S,3S (Scheme 3) or 2R,3R (Scheme 2)9
diasteromeric series of seco-pancratistatin derivatives. Our atten-
tion was first drawn to this discrepancy in contrasting the potent
CYP3A4 inhibitory lycorines8 with potent 2R,3R seco-analogs,9
derivatives that display opposite absolute configuration at C2 and
C3. The present results fully confirm the generality of this remark-
able effect. For example seco-derivatives 32 and 18 both exhibited
a Ki of 0.03 lM, as potent as the clinical antifungal P450 inhibitor
ketoconazole. Likewise, the next potent derivatives in both series
are the C6 tosyl derivatives 36 and 20, that both exhibited Ki values
of 0.07 lM. Reversal of the stereochemistry at C2 and C3 has no
apparent effect on CYP3A4 inhibition, nonetheless we have shown
that C3 must contain a small lipophilic H-bond acceptor. The con-
formational mobility in both sets of potent seco-pancratistatin ana-
log may allow either series to occupy a less rigid portion of the
enzyme active site in the C2/C3 region.
In conclusion, we report a concise, efficient strategy towards the
synthesis of fully functionalized seco-pancratistatin analogs 32–36
bearing the 2S,3S,4S,5S absolute configuration. Rapid entry to this
for 34 (Ki 0.24 lM). The overall SAR data is consistent with our pre-
vious observations in both the lycorane8 and seco-pancratistatin9
series and provides for further insights into essential pharmacolog-
ical elements of the potent CYP3A4 inhibitors 32 and 36.
It is apparent that the 1-benzamide functionality is essential as
compounds 27–31 were inactive and since there is a ten fold differ-
ence in activity between MOM-1-benzamide 32 and MOM-1-car-
bamate 34. This suggests that the C1 part of the molecule must
occupy a fairly bulky, polar pocket within the enzyme active site
with the amide entity possibly functioning as an H-bond acceptor.
Substitution at C6 is seen to be quite sensitive, for example
comparing 32 versus 35 (Table 1) pointing to a moderately sized
lipophilic binding site within the enzyme. Again, these results are
fully in accordance with Ki values recorded for 17 (1.55 lM) and
18 (0.03 l
M)9 (Scheme 2), as well as the pronounced CYP3A4
inhibitory effects of TBS-substituted derivatives of lycorine 5.8
Overall, the introduction of functional groups at C6 other than
TBS had varying effects on inhibitory activity.
The bulky 6-TBDPS group (19, Scheme 2) proved somewhat det-
rimental to CYP3A4 inhibition (Ki 0.58 lM) while introduction of a

2338
J. McNulty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2335–2339
O
O
O
O
HO
OTBS
OTBS
O
O
OH
OiPr
O
O
O
O
O
O
15
a
iPr
O
O
O
26
b
27
11
O
O
O
O
O
O
MOMO
MOMO
MOMO
OTBS
OTBS
OTBS
O
d
c
O
O
O
O
f
O
O
e
OiPr
Br
OH
28
29
30
OH
4
3
HO
OH
O
O
O
O
5
2'
2
MOMO
MOMO
6
3'
O
OTBS
OTBS
1
OH
O
1'
O
O
O
O
HN
6'
5'
g
h
O
4'
N3
1"
HN
6"
5"
2"
3"
31
O
32
4"
33
i
j
O
O
O
O
O
O
MOMO
MOMO
MOMO
OTos
OTBS
OH
O
O
O
O
O
O
k
HN
HN
O
HN
36
35
O
34
O
O
Scheme 3. Synthesis of seco-pancratistatin analogs employing as key step an aldol of the lithium enolate of isopropyl (3,4-methylenedioxyphenyl) acetate. Reagents and
conditions: (a) CH3CHBrCH3, K2CO3, MeCN, reflux, 6 h, 95%; (b) LiHMDS, THF, À78 °C to rt, 2 h, 90%; (c) DIPEA, MOMCl, DCM, 0 °C to rt, 10 h, 96%; (d) LiAlH4, THF, 0 °C to rt, 3 h,
93%; (e) NBS, PPh3, TEA, DCM, 0 °C to rt, 5 h, 88%; (f) NaN3, DMF, rt, 5 h, 93%; (g) (i) 10% Pd/C, H2, THF, rt, 3 h; (ii) BzCl, TEA, DCM, rt, 2 h, 98%; (h) 2 M HCl, THF, rt, 2 h, 89%; (i)
(i) 10% Pd/C, H2, THF, rt, 3 h; (ii) Boc2O, TEA, DCM, rt, 2 h, 98%; (j) TBAF, THF, rt, 1 h, 97%; and (k) TosCl, py, DCM, rt, 5 h, 98%.
Table 1
Inhibitory activity against the biotransformation of 7-benzyloxyquinoline by cDNA-
expressed human CYP3A4
Compound
Ki (
lM)
pKi (M)a
27
29
31
na
na
na
—
—
—
32
33
0.03
na
7.54 ( 0.01)
—
34
35
36
0.24
1.78
0.07
0.03
6.62 ( 0.04)
5.75 ( 0.02)
7.16 ( 0.05)
7.48 ( 0.02)
Ketoconazole
Figure 1. X-ray structure of carbamate 34.
a
Values are means of three experiments, standard deviation is given in paren-
theses (na = not active at 10 M).
l
series was realized via a highly diastereoselective lithium enolate
mediated aldol reaction of phenylacetate 26 with
L
-threose alde-
overall effects of (i), (ii), and (iii) also appear additive in that the
most potent cytochrome inhibitors have both a small, flexible H-
bond acceptor at C3 and lipophilic substituents at C4 and C6. (iv)
There appears no relation of the CYP3A4 inhibitory activity to the
absolute stereochemistry at C2 and C3 in these conformationally
mobile seco-diastereomers. These results clearly point to potential
problems with lipophilic functionalization, particularly at C3, cor-
responding to the C1 position in the natural pancratistatin series.
This area was also highlighted as critical wherein we showed narci-
clasine 7 to be a potent inhibitor, while pancratistatin 6 proved
inactive to CYP3A4. It now appears likely that the polar C1 hydro-
xyl group present in pancratistatin prevents its interaction with
hyde 15. The novel 2S,3S-analogs are in contrast to the correspond-
ing 2R,3R-variants 17–25 previously reported. A more general and
detailed SAR study has evolved, in conjunction with our previous
studies8,9 allowing for the following conclusions to be made for po-
tent CYP3A4 activity. (i) A small H-bond acceptor region at C3 (C1
in the lycorane series8) leads to significant interaction with
CYP3A4. The strongest cytochrome interaction was seen for the
flexible C3 O-MOM group. (ii) A lipophilic substituent at C6 is re-
quired for potent activity. (iii) Bulky lipophilic substitution at C4
(C2 in the lycorane series8) enhances inhibition, indicative of a
large hydrophobic binding pocket in the enzyme active site. The

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this critical metabolizing enzyme and thus contributes signifi-
cantly to the selective anticancer versus P450 interactive activity
observed.
All of the seco-analogs 17–25 and 31–36 reported in Schemes 2
and 3 were found to be devoid of anticancer activity (MCF-7) at
10 lM concentration, in sharp contrast to pancratistatin and narci-
clasine positive controls. These results are in complete agreement
with prior work from various laboratories,2,3 showing that a fully
functionalized, 2,3,4-triol-containing phenanthridone is required
for cystostatic activity.
Lastly, we have identified two potent derivatives 18 and 32 that
exhibit CYP3A4 inhibitory activity at the nanomolar level, as well
as two slightly less potent derivatives 20 and 36. This activity is
similar to the clinical antifungal agent ketoconazole. Extension of
these leads towards the synthesis of novel, selective antifungal
agents are in progress in our laboratories.
Acknowledgments
We thank NSERC and McMaster University for financial support
of this work. We are grateful to Dr. D. Hughes for obtaining high
field NMR data and Dr. J. F. Britten for obtaining the crystallo-
graphic data.
Supplementary data
Supplementary data (CCDC file 654288 contains the supple-
mentary crystallographic data for 34) associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2010.01.157.
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