Progress toward the total synthesis of lucentamycin A: Total synthesis and biological evaluation of 8-epi-lucentamycin A

Article abstract of DOI:10.1021/jo902115s

(Chemical Equation Presented) Synthetic efforts toward the cytotoxic peptides lucentamycins A-D are described that resulted in the total synthesis and biological evaluation of 8-epi-lucentamycin A in 15 steps with 2.2% overall yield. The key epinonproteog

Full text of DOI:10.1021/jo902115s

pubs.acs.org/joc
evaluation seemed warranted.5 Moreover, the lucentamycins
Progress toward the Total Synthesis of Lucentamycin
A: Total Synthesis and Biological Evaluation of
8-epi-Lucentamycin A
were attractive as a target for our Program in the synthesis of
unnatural analogues coupled with biological evaluation and
target elucidation.6-10
R. Nathan Daniels, Bruce J. Melancon, Emily A. Wang,
Brenda C. Crews, Lawrence J. Marnett,
Gary A. Sulikowski, and Craig W. Lindsley*
Departments of Chemistry, Biochemistry, and Pharmacology,
Vanderbilt University, Nashville, Tennessee 37232
craig.lindsley@vanderbilt.edu
Received October 2, 2009
FIGURE 1. Structures of lucentamycin A-D.
The retrosynthesis of lucentamycin A (1) involved clea-
vage of the two amide bonds of the nonproteogenic
3-methyl-4-ethylideneproline nucleus to afford L-leucine
tert-butyl ester 2, the functionalized lysine 3, and unnatural
proline 4 (Scheme 1). The functionalized lysine was envi-
sioned to arise by acylation and guanidation of L-lysine
methyl ester 5. The key nonproteogenic 3-methyl-4-ethyli-
deneproline 6 would be accessed through chiral vinyl ami-
nosulfoxonium salt chemistry as reported by Gais, for which
a single X-ray crystal of 6 was disclosed.11
Synthetic efforts toward the cytotoxic peptides lucenta-
mycins A-D are described that resulted in the total
synthesis and biological evaluation of 8-epi-lucentamycin
A in 15 steps with 2.2% overall yield. The key epi-
nonproteogenic 3-methyl-4-ethylideneproline was syn-
thesized via a titanium-mediated cycloisomerization re-
action.
While synthetic effort was focused on the synthesis of 6, we
initiated a model study en route to an unnatural congener 16
of lucentamycin A, wherein the nonproteogenic 3-methyl-
4-ethylideneproline 6 was replaced with natural L-proline
(Scheme 2) to evaluate potential racemization problems and
to develop structure-activity relationships for the cytotoxi-
city of 1 against HCT-116 cells. Racemization at C16 was not
unexpected as the R-amino group of the lysine was acylated
with a benzoyl group, well documented to form a Leuch
anhydride intermediate, and notorious for promoting race-
mization as the “enol” form is aromatic.12 In the event,
Fmoc-protected L-proline 7 was coupled under HATU con-
ditions with leucine tert-butyl ester 2 to provide the dipeptide
Recently, Fenical and co-workers reported on the isola-
tion and characterization of four novel 3-methyl-4-ethylide-
neproline-containing peptides, lucentamycins A-D, from
the fermentation broth of a marine-derived actinomycete
identified as Nocardiopsis lucentensis (Figure 1).1 Impor-
tantly, lucentamycins A and B displayed significant in
vitro cytotoxicity, IC50 values of 0.2 and 11 μM, res-
pectively, against HCT-116 human colon carcinoma.1
On the basis of the biological activity of lucentamycin A
and the broad spectrum of biological activity (antibiotic,
antifungal, anticancer) of other agents derived from
Nocardiopsis,1-4 a total synthesis campaign targeting lucen-
tamycin A to provide sufficient material for biological
(6) Kennedy, J. P.; Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett.
2009, 19, 3304–3308.
(7) Lewis, J. A.; Daniels, N. R.; Lindsley, C. W. Org. Lett. 2008, 10, 4545–
4548.
(8) Daniels, N. R.; Fadeyi, O.; Lindsley, C. W. Org. Lett. 2008, 10, 4097–4050.
(9) Fadeyi, O.; Lindsley, C. W. Org. Lett. 2009, 11, 943–945.
(10) Fadeyi, O.; Daniels, R. N.; DeGuire, S.; Lindsley, C. W. Tetrahedron
Lett. 2009, 50, 3084–3087.
(11) Tiwari, S. K.; Gais, H.-J.; Lindenmaier, A.; Babu, G. S.; Raabe, G.;
Reddy, L. R.; Kohler, F.; Gunter, M.; Koep, S.; Iska, V. B. R. J. Am. Chem.
Soc. 2006, 128, 7360–7373.
(1) Cho, J.; Williams, P. G.; Kwon, H. C.; Jensen, P. R.; Fenical, W.
J. Nat. Prod. 2007, 70, 1321–1328.
(2) Ayuso, A.; Clark, D.; Gonzalez, I.; Salazar, O.; Anderson, A.;
Genilloud, O. Appl. Microbiol. Biotechnol. 2005, 67, 795–806.
(3) Bergy, M. E. J. Antibiot. 1968, 21, 454–457.
(4) Dolak, L. A.; Castle, T. M.; Laborde, A. L. J. Antibiot. 1980, 33, 690–
694.
(5) Kennedy, J. P.; Brogan, J. T.; Lindsley, C. W. J. Nat. Prod. 2008, 71,
1783–1786.
(12) Siemon, I. Z.; Konopinska, D. Rocz. Chem. 1970, 44, 785–791.
8852 J. Org. Chem. 2009, 74, 8852–8855
Published on Web 10/29/2009
DOI: 10.1021/jo902115s
r
2009 American Chemical Society

Daniels et al.
JOCNote
SCHEME 1. Retrosynthesis of Lucentamycin A (1)
SCHEME 2. Lucentamycin A Model Study To Deliver 16
8 in 99% yield. Fmoc deprotection with 5% piperidine in
DMF delivered the dipeptide 9 in quantitative yield. L-Lysine
methyl ester 5 was treated with 10 to provide the bis-N-Boc-
protected arginine derivative 11 in 77% yield.13 Acylation
with benzoyl chloride and hydrolysis employing LiOH af-
forded 13 in 62% yield over the two steps. The coupling of 9
to 13 justified the model study, as our initial EDCI/HOBt/
collidine coupling conditions generated >90% chemical
yield, but a 39:61 ratio of the desired 14 to the epimerized
15. The degree of racemization was determined by analytical
LCMS and confirmed by 1H NMR. At this point, we
evaluated a variety of coupling reagents, additives, and
solvent/temperature conditions. Ultimately, the conditions
of HATU and collidine without HOAt proved optimal,
generating a 92:8 ratio of 14:15 in yields exceeding 90%
and readily separable by column chromatography. A final
global deprotection with 10% TFA in DCM afforded the
unnatural analogue 16 of lucentamycin A in 60% yield.
Attention now turned to the construction of the key
nonproteogenic 3-methyl-4-ethylideneproline 4. Application
of the Gais protocol proved arduous, with difficult E/Z
mixtures at multiple points along the 11-step sequence,
which ultimately result in complex chromatographic separa-
tions where even sophisticated reverse phase systems failed
to deliver key intermediates in yields satisfactory for carrying
forward en route to a total synthesis of 1. At this point, we
revised our retrosynthesis for 4, and we envisioned access
to 4 by a titanium-mediated cycloisomerization reaction14
(Scheme 3) to afford 17, which would be derived from
Garner’s aldehyde 18. Key to the success of this route would
be the ability to epimerize the R-carbon; however, this route
would also enable the synthesis of the C8 epimer of lucenta-
mycin A, 8-epi-lucentamycin A, and further construct SAR.
In the event, Garner’s aldehyde 18 smoothly undergoes a
Wittig reaction providing 19 in 83% yield.15 Deprotection
with p-TsOH in MeOH affords 20, which is then converted to
SCHEME 3. Revised Retrosynthesis of 4
the corresponding oxazolidin-2-one 21 in 83% yield for the
two steps. Deprotonation with KHMDS and alkylation with
mesylate 22 delivers 23. A titanium-mediated cycloisomeri-
zation reaction delivers bicycle 17, which sets the methyl
stereocenter and the relative regiochemistry of the 3-methyl-
4-ethylideneproline nucleus in 91% yield.14 Detailed NOE
studies and historical accounts confirmed the stereochemical
assignment.16 Opening of the cyclic carbamate with methox-
ide generates 24, which is oxidized to the aldehyde 25 in 98%
yield. Finally, a buffered bleach oxidation of the aldehyde
leads to 26, the epimer of the key nonproteogenic 3-methyl-4-
ethylideneproline 4, in 60% yield. A double deprotonation/
kinetic quench with AcOH failed to provide the desired 27
(13) Crane, C. M.; Boger, D. L. J. Med. Chem. 2009, 52, 1471–1476.
(14) Yang, X.; Zhai, H.; Li, Z. Org. Lett. 2008, 10, 2457–2460.
(15) Lebel, H.; Paquet, V.; Proulx, C. Angew. Chem., Int. Ed. 2001, 40,
2887–2890.
(16) See the Supporting Information for full details.
J. Org. Chem. Vol. 74, No. 22, 2009 8853

Daniels et al.
JOCNote
SCHEME 4. Synthesis of an Epimeric Non-Proteogenic
3-Methyl-4-ethylideneproline 26 and Attempts To Epimerize
To Deliver 27
SCHEME 5. Synthesis of the Key Non-Proteogenic 3-Methyl-
4-ethylideneproline 30 and Southern Dipeptide Fragment 31
SCHEME 6. Total Synthesis of 8-epi-Lucentamycin A (33)
(Scheme 4), affording only starting material 26 with no
evidence of any epimerization. Alternative approaches to
epimerize 25, 26, and esters of 26 met with similar unpro-
ductive results.17 Thus, our strategy adjusted to target the
synthesis and biological evaluation of 8-epi-lucentamycin A
employing 26. However, removal of the methyl carbamate in
26 proved equally challenging, and we were unable to affect
this key transformation under a variety of reaction condi-
tions.
Therefore, we again modified our approach for the total
synthesis of 8-epi-lucentamycin A. As shown in Scheme 5,
bicycle 17 was treated in a single pot with 3.0 M NaOH under
microwave irradiation, followed by an in situ protection of
the secondary amine to deliver Boc-protected pyrrolidine 28
in 80% yield.18 Alcohol 28 smoothly underwent oxidation to
the corresponding aldehyde 29 in 70% yield. Repetition of
the buffered bleach oxidation of 29 provides 30, the epimer of
the key nonproteogenic 4. The reaction to deliver acid 30
proceeded cleanly, therefore crude material (>95% pure)
was carried forward into the coupling step. Thus, crude 30
was directly coupled to L-leucine tert-butyl ester 2 under
HATU conditions (61% for two steps), followed by a
chemoselective deprotection of the Boc group under anhy-
drous acidic conditions to deliver 31 in 68% yield.19
Dipeptide 31 and 13 were treated with our optimal cou-
pling system of HATU and collidine in DMF/DCM at 0 °C
to deliver 32 in 81% yield, as a 92:8 ratio of diastereomers.
After separation of the racemized material, a global depro-
tection employing 25% TFA in DCM provided 8-epi-lucen-
tamycin A 33 in an unoptimized 47% yield (Scheme 6).
NMR data of the epimeric lucentamycin A 33 agreed well
with the natural product 1, with the expected exceptions due
to the epimerization of C8.
Lucentamycin A (1) displayed significant in vitro cyto-
toxicity, IC50 value of 0.2 μM, against HCT-116 human
colon carcinoma. With 8-epi-lucentamycin A (33) in hand,
we evaluated its affect on an HCT-116 cell line in order to
determine if the stereochemistry of the nonproteogenic
3-methyl-4-ethylideneproline nucelus was critical for biolo-
gical activity. Thus, a standard 48 h cell viability assay was
performed with 16 and 33 at six concentrations (0, 0.025, 0.1,
0.4, 2.0, and 10 μM) relative to podophyllotoxin as a positive
control.20 The control performed as expected, providing an
IC50 value of 0.03 μM. However, neither unnatural, epimeric
Lucentamycin A (33) nor 16 had an effect on HCT-116 cell
viability up to 10 μM. The cytotoxicity data generated with
both 33 and 16 suggest that the stereochemistry at C8, and
the topology afforded by the natural product, is essential for
potent in vitro cytotoxicity (IC50 =0.2 μM) of lucentamycin
A (1) against HCT-116 cells.
(17) Clark, D. L.; Chou, W. N.; White, J. B. J. Org. Chem. 1990, 55, 3975–
3977.
(18) Hanessian, S.; Ninkovic, S. J. Org. Chem. 1996, 61, 5418–5424.
(19) Gibson, F. S.; Bergmeier, S. C.; Rapoport, H. J. Org. Chem. 1994, 59,
3216–3218.
(20) Lear, Y.; Durst, T. Can. J. Chem. 1996, 74, 1704–1708.
8854 J. Org. Chem. Vol. 74, No. 22, 2009

Daniels et al.
JOCNote
Thus, synthetic efforts toward the lucentamycins A-D,
1-4, have been reported culminating in the total synthesis of
8-epi-lucentamycin A (33). The synthesis features a titanium-
mediated cycloisomerization reaction to construct the key,
epimeric nonproteogenic 3-methyl-4-ethylideneproline 30.
The convergent synthetic route afforded 8-epi-lucentamycin
A 33 in 15 steps, with a 10 step longest linear sequence, and
an overall yield of 2.2%. Biological evaluation of 8-epi-
lucentamycin A (33) and another unnatural congener 16
indicated that both were inactive relative to natural 1, with
an IC50 value of >10 μM in a HCT-116 human carcinoma
cell line. Interestingly, these studies suggest that the natural
configuration of the nonproteogenic 3-methyl-4-ethylidene-
proline 4 is essential for bioactivity. Future efforts are
focused on developing chemistry to access the natural,
nonproteogenic 4 and the total synthesis and biological
evaluation of lucentamycin A (1), as there are not sufficient
quantities of 1 for biological evaluation.
added immediately followed by collidine (10.6 μL, 0.080 mmol).
The reaction was stirred at 0 °C for 6 h and then diluted with DCM
(4 mL) and washed with H2O (3 ꢀ 2 mL) and filtered through a
phase separator. The crude mixture was concentrated in vacuo and
purified by flash chromatography (1:0 to 1:1 Hex:EtOAc) to yield
the product as a crusty foam (47.0 mg, 0.059 mmol) in 80.5% yield.
[R]20D 23.9 (c 0.2, CHCl3); IR (thin film) 3330, 2964, 2929, 2929,
1722, 1639, 1154, 1135 cm-1 1H NMR (600.1 MHz, CDCl3)
;
δ (ppm) 11.47 (br s, 1H), 8.35 (br s, 1H), 7.82 (m, 2H), 7.48 (t, J=
7.4 Hz, 1H), 7.40 (t, J=7.7 Hz, 2H), 7.11 (d, J=8.5 Hz, 1H), 6.94
(br s, 1H), 5.44 (m, 1H), 4.83 (q, J = 6.8 Hz, 1H), 4.49 (d, J =
14.3 Hz, 1H), 4.42 (m, 1H), 4.37 (d, J=1.4 Hz, 1H), 4.30 (d, J=
14.3 Hz, 1H), 3.43 (m, 2H), 3.14 (q, J=7.0 Hz, 1H), 1.93 (m, 1H),
1.86 (m, 1H), 1.67 (m, 2H), 1.62 (d, J=6.8 Hz, 3H), 1.59 (m, 1H),
1.52 (m, 4H), 1.48 (s, 9H), 1.47 (s, 9H), 1.36 (s, 9H), 1.16 (d, J=
7.2 Hz, 3H), 0.82 (d, J=6.1 Hz, 3H), 0.70 (d, J=6.1 Hz, 3H); 13
C
NMR (150.9 MHz, CDCl3) δ (ppm) 171.81, 171.76, 169.8, 167.5,
163.3, 156.1, 153.2, 139.0, 133.3, 131.8, 128.5, 127.3, 118.1, 83.2,
81.2, 79.5, 66.6, 51.7, 51.2, 47.8, 41.2, 40.9, 40.6, 31.7, 28.9, 28.2,
28.0, 27.9, 24.6, 22.9, 22.6, 21.8, 21.2, 14.7; HRMS (TOF, ESþ)
C42H67N6O9 [M þ H]þ calcd 799.4970, found 799.4970.
Experimental Section
8-epi-Lucentamycin A (33). 32 (38.3 mg, 0.048 mmol) was
dissolved in anhydrous DCM (0.38 mL) and cooled to 0 °C.
TFA (0.12 mL) was added dropwise and the solution was
allowed to warm to room temperature over 3 h and stirred
overnight under an argon atmosphere. MeOH was added to the
reaction mixture, which was then concentrated in vacuo. More
MeOH was added and the reaction was concentrated again.
Because of the high polarity of the compound, the product was
purified by reverse phase preparatory HPLC to yield the
product as a white solid (12.3 mg, 0.023 mmol) in 47.3% yield.
[R]20D 28.5 (c 0.2, MeOH); 1H NMR (600.1 MHz, DMSO-d6) δ
(ppm) 10.19 (m, 1H), 8.63 (d, J = 7.1 Hz, 1H), 8.22 (d, J =
9.0 Hz, 1H), 7.90 (m, 2H), 7.51 (t, J =7.3 Hz, 1H), 7.44 (t, J=
7.6 Hz, 2H), 6.95 (m, 2H), 5.26 (m, 1H), 4.69 (d, J=1.7 Hz, 1H),
4.45 (m, 1H), 4.16 (d, J=16.2 Hz, 1H), 4.00 (m, 1H), 3.89 (d, J=
16.5 Hz, 1H), 3.32 (br s, 2H), 3.05 (m, 1H), 2.93 (m, 1H), 2.62 (m,
1H), 1.65 (m, 1H), 1.55 (m, 7H), 1.47 (m, 1H), 1.39 (m, 1H), 1.32
(m, 2H), 1.16 (d, J = 7.0 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H),
0.80 (d, J = 6.1 Hz, 3H); 13C NMR (150.9 MHz, DMSO-d6) δ
(ppm) 177.0, 171.3, 170.5, 166.3, 157.6, 140.4, 133.8, 131.3,
128.1, 127.6, 115.0, 66.5, 52.3, 51.4, 47.9, 45.1, 41.3, 40.6, 40.0,
30.1, 28.1, 24.9, 23.2, 21.6, 21.2, 14.3; HRMS (TOF, ESþ)
C28H43N6O5 [M þ H]þ calcd 543.3295, found 543.3295.
(S)-tert-Butyl 2-((2R,3R,Z)-4-Ethylidene-3-methylpyrrolidine-
2-carboxamido)-4-methylpentanoate (31). Boc-protected 31
(50.8 mg, 0.12 mmol) was added to a small vial and placed
under argon. Anhydrous EtOAc (0.45 mL) and anhydrous HCl
in dioxane (4 M, 0.15 mL, 0.6 mmol) were then added and
the reaction was stirred for 1 h. Additional anhydrous EtOAc
(0.45 mL) and anhydrous HCl in dioxane (4 M, 0.15 mL,
0.6 mmol) were then added at 1, 2, and 4 h total reaction time
for a total of four injections of each of EtOAc and HCl in
dioxane. NaOH (1 N) was added to neutralize the reaction. The
aqueous layer was separated and extracted with EtOAc (3 ꢀ
5 mL). The combined organic layers were dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
flash chromatography (1:0 to 19:1 DCM:MeOH) to yield the
product as an oil (26.2 mg, 0.081 mmol) in 67.5% yield. [R]20
D
29.7 (c 0.2, CHCl3); Rf 0.52 (9:1, DCM:MeOH); IR (thin film)
3331, 2960, 2929, 2870, 1735, 1673, 1509, 1368, 1151 cm-1; 1H
NMR (600.1 MHz, CDCl3) δ (ppm) 7.58 (d, J = 8.5 Hz, 1H),
5.24 (m, 1H), 4.47 (td, J=8.9, 5.0 Hz, 1H), 3.64 (s, 2H), 3.25 (d,
J=7.5 Hz, 1H), 2.63 (m, 1H), 2.36 (br s, 1H), 1.62 (m, 1H), 1.56
(d, J = 6.5 Hz, 3H), 1.52 (m, 1H), 1.43 (s, 9H), 1.19 (d, J = 6.8
Hz, 3H), 0.93 (d, J = 6.2 Hz, 6H); 13C NMR (150.9 MHz,
CDCl3) δ (ppm) 173.1, 172.1, 143.8, 114.3, 81.5, 68.3, 50.8, 47.8,
42.7, 41.7, 27.9, 25.0, 22.9, 21.9, 17.7, 14.5; HRMS (TOF, ESþ)
C18H33N2O3 [M þ H]þ calcd 325.2491, found 325.2491.
Acknowledgment. We are very grateful to Professor
Fenical (UCSD) for NMR spectra of lucentamycins A-D.
This work was supported, in part, by the Department of
Pharamacology, Vanderbilt University, and the National
Institutes of Health 5R01CA059515 (G.A.S.).
(S)-tert-Butyl 2-((2R,3R,4Z)-1-((S)-2-Benzamido-6-(2,3-bis-
(tert-butoxycarbonyl)guanidino)hexanoyl)-4-ethylidene-3-methyl-
pyrrolidine-2-carboxamido)-4-methylpentanoate (32). 31 (23.7 mg,
0.073 mmol) and 13 (36.0 mg, 0.073 mmol), in anhydrous DCM
(0.3 mL each), were added to a flame-dried flask under argon via
syringe. Anhydrous DMF (0.3 mL) was added and the solution
was cooled to 0 °C. O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetra-
methyluronium hexafluorophosphate (41.7 mg, 0.111 mmol) was
Supporting Information Available: Experimental proce-
dures, characterization data, and 1H and 13C NMR spectra
for all new compounds, 8-33. This material is available free of
charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 22, 2009 8855