1
The Convergent Synthesis of the Anti-cancer Agent Geralcin B
William Brew, Cameron Incognito, Timothy Saunders, Michael Stetler
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802
web5052@psu.edu, cpi5006@psu.edu, tps153@psu.edu, mjs5526@psu.edu
Chem 431W
12/12/2012
Abstract
The first chemical synthesis of the anti-cancer agent Geralcin B (2), an α,β-unsaturated γlactono-hydrazide, is described. A theoretical synthetic scheme involving the formation of an
α,β-unsaturated γ-lactone 3, and its subsequent coupling with a cis-alkenyl acethydrazide species
16 to form Geralcin B (2), was developed. The initial steps of the alkenyl acethydrazide were
successfully synthesized; specifically, the α,β-unsaturated carboxylic acid 12 was synthesized
through a Knoevenagel condensation, followed by the bromination of the alkene, and finally, the
formation of a cis-bromoalkene 13.
Introduction
Hydrazine and hydrazide compounds are commonly used in clinical therapeutic
antibiotics.1 There are, however, only four known hydrazide compounds that have been found in
nature: hydrazidomycins A, B, C, and montamine.1 These compounds have unknown biological
roles and varying cytotoxic properties against cancer cells: for instance, montamine has an IC50,
or half maximal inhibitory concentration, of 43.9 μM, while hydrazidomycins A through C have
an average IC50 of 0.37 μM.1 Two new alkyl-hydrazides, Geralcin A (1) and B (2), seen in Figure
1, were recently isolated in 2012 from the actinobacteria Streptomyces sp. LMA-545.1 It has
been found that Geralcin B (2) is cytotoxic against MDA231 breast cancer cells with an IC50 of 5
2
μM, making it a possible anticancer drug component.1 Both alkyl hydrazides 1 and 2 can be
formed from the nucleophilic substitution of an alkyl-hydrazide and the α,β-unsaturated γlactone 3-(5-oxo-2H-furan-4-yl)-propanoyl chloride 3.1
Figure 1: Structures of Geralcin A (1) and B (2)
Figure 1: Two isolated alkyl-hydrazide species, Geralcin A (1) and the desired product Geralcin B (2).
Figure 2: Structure of 3-(5-oxo-2H-furan-4-yl)-propanyl chloride (3)
3
Figure 2: A reactant in the nucleophilic substitution reaction involving the alkyl-hydrazide to produce the product
Geralcin B (2).
3
This experiment was the first attempted total synthesis of Geralcin B (2). Le Goff et al.
propose a hypothetical biosynthetic pathway to form the compound, using glycoaldehyde and an
acyl carrier protein.1 The two are first condensed to glutaric acid in a process catalyzed by an
AfsA-like protein.1 An aldol condensation followed by an acyl substitution with the hydrazide
catalyzed by a dehydratase would afford the product, as shown in Figure 3.1
Figure 3: Hypothetical Geralcin B Synthesis
Figure 3: A proposed synthetic route from Le Goff et al. for Geralcin B (2) from glycoaldehyde and an acyl carrier
protein.
Our group plans to form a cis-alkenyl acethydrazide, and use a substituted lactone that is
nearly identical to what Le Goff et al. propose in order to perform an acyl substitution.1 We
propose a synthesis of Geralcin B (2) through a convergent synthetic route ultimately ending in a
nucleophilic acyl substitution reaction between an alkenyl acethydrazide 4 and an α,βunsaturated γ-lactone 3, as shown in Figure 4. All starting materials and catalysts were carefully
chosen to be commercially available as well as fiscally practical. The syntheses of 3 and 4 can
be carried out simultaneously, reducing the overall product synthesis time. A retrosynthetic
analysis of Geralcin B (2) is detailed in Scheme 1.
4
Scheme 1: Retrosynthesis of Geralcin B (2)
Figure 4: Geralcin B (2) can be formed from the condensation of an alkenyl acethydrazide 4 and the α,β-unsaturated
γ-lactone, 3-(5-oxo-2H-furan-4-yl)-propanyl chloride (3).
The formation of the α,β-unsaturated γ-lactone 3 would start with the cyclization of 2phenylthioacetic acid (5) via LDA in THF-ether followed by mCPBA in CH2Cl2 to form the
butyrolactone 6, as demonstrated by Iwai et al.2 Desaturation of the lactone ring and substitution
of the thionyl group on butyrolactone 6 has been shown by Reichelt to occur via a Michael
addition when exposed to sodium hydride and the corresponding conjugated 2-propanoic acid
ester species to form 2,5-dihydro-2-oxo-3-furanpropanoic acid ethyl ester 7.3 Kawabe et al. then
demonstrated that the ester 7 can then be hydrated in an aqueous solution of hydrochloric acid to
form 2,5-dihydro-2-oxo-3-furanpropanoic acid (8).4 The acyl chloride 3 would then be formed
via the nucleophilic acyl substitution reaction of oxalyl chloride and the carboxylic acid 8
according to Wang et al.5 Synthetic conditions can be seen in Scheme 2.
5
Scheme 2: Synthesis of α,β-unsaturated γ-lactone 3
The addition of malonic acid (9) and pentanal (10) in THF (1M) to pyrrolidine and
pyridine has been shown to yield the carboxylic acid 11.6 Bromination in dichloromethane at
room temperature has been demonstrated by Feutren et al. to yield the dibromo-carboxylic acid
12.7 Formation of the cis-bromoalkene 13 would then proceed by heating a mixture of 12 and
sodium bicarbonate in DMF at reduced pressure.8 Adding catalytic copper iodide to a mixture of
hydrazine (14), potassium carbonate, and dimethylethylenediamine in toluene under heat should
yield the cis-alkenyl hydrazide 15.9 Protection of the secondary amine has been demonstrated
6
via formation of the imine 16 by refluxing 15 and benzaldehyde in toluene, followed by the
addition of (BOC)2O (BOC - tert-butyloxycarbonyl) at room temperature.10 Conversion of the
imine functionality to the desired primary amine proceeds through the addition of 16 to a
solution of potassium bisulfate and water, and stirring.10 Column chromatography will be used to
separate the desired precursor from the side products, and sodium hydroxide in water added to
yield 17.10 Functionalization of 17 with acetyl chloride proceeds via nucleophilic acyl
substitution in a solution of sodium bicarbonate, water and acetone to yield 18.11 Deprotection
of the amine would occur via the removal of the BOC protecting group using TFA in
dichloromethane to give the alkenyl acethydrazide 4.12 Synthetic conditions can be seen in
Scheme 3.
7
Scheme 3: Synthesis of alkenyl acethydrazide 4
The final product 2 would then be afforded by the addition of 3 to 4 via nucleophilic acyl
substitution, in a solution of sodium bicarbonate, water, and acetone.11 Synthetic conditions can
be seen in Scheme 4.
8
Scheme 4: Convergent Synthesis of Geralcin B (2)
When approaching the design of the total synthesis of Geralcin B (2), key intermediates
had to be considered. Formation of the cis-alkenyl acethydrazide 15 would be difficult due to
the energetically-unfavorable cis configuration of the bromide 13, the instability of which is
emphasized in the literature8 by the description of the product as “highly volatile.” The formation
of the cis-alkenyl acethydrazide 15 from 13 was also concerning due to the sp2 hybridization of
the nitrogen species, so the reaction must proceed through a metal-catalyzed substitution of the
halogenated alkene 13. Another concern was the coupling of the cis-alkenyl
acethydrazide 17 with acetyl chloride to ultimately form the hydrazide-bridged species 4. In
order to properly guide the acyl substitution with the primary amine on 15, a protecting group on
the secondary amine is necessary.
Results and Discussion
The α,β-unsaturated carboxylic acid 11 was prepared in a 47.2% yield (literature6 yield of
77%) from malonic acid (9) and pentanal (10), from which the bromination of the alkene yielded
the dibromo-carboxylic acid 12 in a 19.0% yield (literature7 yield of 96%). Finally, the cisbromoalkene 13 was formed in a trace yield (literature8 yield of 87%). Each product was
characterized by 1H and 13C NMR as well as infrared spectroscopy.
9
Scheme 5: Synthesis of cis-bromoalkene 13
Synthesis and Characterization
The three initial steps of the alkenyl acethydrazide component formation were
successfully synthesized in poor yield. Due to time constraints it was not possible to complete
other trials for the third step which may have been helpful for improving reaction conditions and
possibly the reaction yield.
The original procedure for the synthesis of the α,β-unsaturated carboxylic acid 11
described by Kemme et al. called for the stirring of the reaction mixture at 10oC overnight.6 Due
to lab access restrictions, the temperature was unable to be precisely controlled. Several trials
were conducted in order to determine the most successful reaction conditions: two samples were
stirred for 48 hours, two at room temperature and two at 0 oC. The latter reaction conditions
proved to be marginally more successful with a 47.2% yield. Co-elution of the intended product
11 and starting materials during column chromatography prevented complete recovery of the
product.
The extraction methods described by Feutren et al. for isolation of the dibromoalkene 12
were also modified.7 A significant amount of product was lost during extraction due to
10
inadequate volume of dichloromethane, which prevented the product from transferring to the
organic phase. For the second trial, an excess of dichloromethane was added to the crude
mixture before neutralization of Br2 with sodium thiosulfate and brine, allowing for a better
phase transfer of the pure product 12.
Improper mediation of the gas evolution during the formation of 13 may have caused an
equilibrium shift that may have affected the reaction rate. Imprecise vacuum control may also
have caused the product to be removed with the gases evolved as byproducts of the reaction
before they had time to condense in the cold finger. This, along with improper heat mediation,
could be the cause of the low yield.
Carboxylic acid 11 was synthesized and isolated in a 47.2% yield as a colorless oil, which
was characterized by 400 MHz 1H NMR (Appendix 1). The most downfield peak present is that
of a singlet carboxylic acid proton at ~11.5 ppm (1H). The β and α alkene protons are observed
as a double of triplets at 7.26 – 7.05 ppm and a doublet at 5.84 – 5.80 ppm (2 x 1H) respectively.
Lastly, the alkyl protons are located in three regions: a triplet of doublets at 2.27 – 2.21 ppm (2H)
corresponding to the protons on carbon 4; a multiplet at 1.50 – 1.31 ppm (6H) corresponding to
the protons on carbons 5, 6, and 7; and lastly the three terminal methyl group protons located at
0.93 – 0.85 ppm as a triplet. The product was also characterized by FT-IR (Appendix 2)
showing the representative alcohol O-H stretch at 2958.5 cm-1, the conjugated carbonyl C=O
stretch at 1694.5 cm-1, the C=C alkene stretch at 1649.6 cm-1, and the C-O single bond stretch for
an acid at 1284.4 cm-1.
The dibromo-carboxylic acid 12 was synthesized and isolated in a 19.0% yield as an
orange-yellow oil, which was characterized by 400 MHz 1H NMR (Appendix 3). The presence
of a carboxylic acid proton is found as a singlet at 11.8822 ppm (1H). The α and β protons,
11
however, are shifted significantly upfield due to the presence of the two bromine substituents and
can be found as a doublet at 4.46-4.43 ppm and a multiplet at 4.38-4.33 ppm (2 x 1H)
corresponding to the protons on carbons 2 and 3, respectively. Lastly, the alkyl protons can be
found in four different peaks, each of which are multiplets: 2.29-2.31 ppm corresponding to
single proton attached to carbon 3, 1.87-1.79 ppm corresponding to the second proton attached to
carbon 3, 1.60-1.33 ppm (4H) corresponding to the four protons attached to carbons 5 and 6, and
0.98-0.93 ppm (3H) corresponding to the terminal methyl protons. The 13C NMR (Appendix 4)
indicates the presence of seven different carbon atoms: a carbonyl carbon atom at 173.8925 ppm,
the two bromine-substituted carbons at 53.45 and 47.40 ppm, and the four alkyl carbon atoms
located from 34.63 to 13.87 ppm. Lastly, characterization by FT-IR (Appendix 5) showed peaks
of an O-H stretch of an alcohol at 2957.4 cm-1, C=O carbonyl stretch at 1719.0 cm-1, and a C-O
single bond for an acid at 1282.7 cm-1.
The cis-bromoalkene 13 was synthesized in a trace yield as an impure colorless oil,
which was characterized by 1H NMR (Appendices 6 and 7). The presence of cis-alkene protons
are evident at 6.15-6.11 ppm and 6.10-6.06 ppm (2 x 1H, t, J=6.92 Hz). The trans-E2
elimination reaction, resulting in the cis configuration of the bromoalkene, was confirmed by the
J-coupling value, which is relatively low compared to the corresponding trans configuration
coupling values. The alkyl protons can be found in four peaks: 2.23-2.19 ppm (2H) as a
multiplet corresponding to the protons attached to carbon 3, 1.41-1.34 ppm (2H) as a multiplet
corresponding to the carbon 4 protons, 1.28-1.19 ppm (2H) as a doublet of triplets corresponding
to carbon 5 protons; and lastly 0.94-0.90 ppm (3H) corresponding to the terminal methyl protons.
The 13C NMR (Appendix 8) shows the bromine substituted sp2 carbon atom at 135.05 ppm, the
second alkene carbon atom at 107.53 ppm, and the four alkyl carbons at 30.30, 29.42, 22.23, and
12
13.90 ppm in increasing numerical order. The remaining peaks in both the 1H and 13C NMR
spectra are a combination of residual solvent impurities and starting materials from the extraction
and the two previous reactions. Lastly, 13 was characterized using gas chromatography
(Appendix 9), which showed a single peak with a retention time of 8.33 minutes, corresponding
to the cis-configuration of bromoalkene 13.
Conclusion
The syntheses of the carboxylic acid 11, the dibromo-carboxylic acid 12, and the cisbromoalkene 13 were successfully but inefficiently synthesized. Several procedural
modifications, including isolation and purification methods, were tested in an attempt to improve
the efficiency of these steps, but were ineffective.
The next step in exploring the synthesis of Geralcin B (2) is to complete the formation of
the cis-alkenyl acethydrazide 4, begin the synthesis of α,β-unsaturated γ-lactone 3, and ultimately
couple 3 and 4 together to produce the anti-cancer agent Geralcin B (2).
Experimental
All chemicals and solvents were ordered from Sigma Aldrich (Milwaukee, WI) and Alfa
Aesar (Ward Hill, MA) and were used as supplied. The 1H and 13C NMR spectra were recorded
on Bruker AVANCE 400 MHz multinuclear NMR spectrometer using CDCl3 as solvent. The
infrared spectra were recorded on a Thermo Nicolet 380 IR spectrometer with diamond ATR.
Gas chromatography was carried out on a Hewlett Packard 5890 Gas Chromatograph using a
13
30m x 0.25 mm I.D. capillary column with a 25 micron coating of 5% phenyl/95% methyl
silicone which was programmed from 40 to 250°C at 4°/min.
2-Heptanoic acid (11). Catalytic pyrrolidine (4.00 μL, 50.0 μmol) and pyridine (809 μL, 10.0
mmol) were added to a solution of malonic acid (9) (520 mg, 5.00 mmol) and pentanal (10) (0.53
mL, 5.00 mmol) in THF (5.00 mL, 1M) at 0oC and stirred overnight. The product was extracted
with ethyl acetate, which was collected and dried over MgSO4. The crude product was then
purified via column chromatography to yield 0.4073 g (47.2%) of 2-Heptanoic acid (11) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ ~11.5 (s, 1H), 7.1315-7.0577 (dt, 1H), 5.84935.8031 (d, 1H), 2.2682-2.2107 (td, 2H), 1.4970-1.3064 (m, 6H), 0.9341-0.8504 (t, 3H); IR (neat)
2958.5, 2928.9, 1694.5, 1649.6, 1416.7, 1284.4 cm-1.
2,3-Dibromoheptenoic acid (12). Bromine (0.18 mL, 3.47 mmol) was added to a solution of 11
(0.868 g, 6.9 mmol) in dry CH2Cl2 (10 mL) and left to stir overnight at room temperature. The
reaction mixture was washed with sodium thiosulfate (2x10 mL, 1M) and brine (2x10 mL), dried
over MgSO4, and condensed to yield 0.178 g (19.0%) of 2,3-dibromoheptenoic acid (12) as an
orange-yellow oil. 1H NMR (400 MHz, CDCl3) δ 11.822 (s, 1H), 4.4588-4.4310 (d, 1H),
4.3844-4.3283 (m, 1H), 2.2894-2.2135 (m, 1H), 1.8704-1.7863 (m, 1H), 1.6036-1.3252 (m, 4H),
0.9833-0.9345 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 173.8925, 51.8647, 47.4047, 34.6260,
28.2806, 21.8635, 13.8715; IR (neat) 2957.4, 2930.6, 2862.2, 1719.0, 1427.8, 1282.7 cm-1.
cis-1-Bromo-1-hexene (13). A mixture of 12 (0.962 g, 3.34 mmol) in DMF (0.716 mL) was
added drop wise over an hour to a solution of NaHCO3 (0.286 g, 3.37 mmol, 1.7 eq.) in DMF
(1.43 mL) heated to 70oC at reduced pressure (97.5082 mmHg). The product was distilled out of
solution and collected via a cold trap (-78oC) to yield 0.962 g (trace yield) of cis-1-bromo-1-
14
hexene (13) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.1512-6.1125 (t, 1H), 6.09586.0615 (t, 1H), 2.2282-2.1923 (m, 2H), 1.4091-1.3412 (m, 2H), 1.2788-1.1933 (dt, 2H), 0.93590.9003 (t, 3H); 13C NMR (100 MHz, CDCl3) δ 135.0545, 107.5316, 30.2951, 29.4199, 22.6222,
13.9041; GC (RT) 8.33 min (100%).
Acknowledgements
We would like to thank Anthony, Jerry, and Ryan for their availability and help in
providing advice and guidance in the laboratory. The help that we received in interpreting
spectra and different issues that arose during laboratory was greatly appreciated, as was the
support of Dr. Katherine Masters. We would also like to thank the stock room employees for
their help in obtaining all necessary chemicals and equipment for this laboratory experiment.
15
References
1.
Le Goff, G.; Martin, M.; Servy, C.; Cortial, S.; Lopes, P.; Bialecki, A.; Smadja, J.;
Ouazzani, J. J. Nat. Prod. 2012, 75, 915-919.
2.
Iwai, K.; Kosugi, H.; Kawai, M. Bull. Chem. Soc. Jpn. 1977, 50, 242-247.
3.
Reichelt, A.; Bur, S. K.; Martin, S. F. Tetrahedron. 2002, 58, 6323-6328.
4.
Kawabe; Takumi; Ishagaki; Machiyo; Sato; Takuji; Yamamoto; Sayaka; Hasegawa;
Yoko. (CanBas Co., Ltd., Japan). Preparation of pyridinylaminopyrrolinediones
and related compounds for treatment of cell proliferation disorders. U.S. Patent
20080275057, 06 November 06, 2008.
5.
Wang, W.; Li, L.; Niu, Y.; Li, S.; Ma, J.; Li, L.; Liu, Y.; Cai, L.; Zhao, W. Bioorg. Med.
Chem. Lett. 2011, 21, 5041-5044.
6.
Kemme, S. T.; Šmejkal, T.; Breit, B. Chem. Eur. J. 2010, 16, 3423-3433.
7.
Feutren, S.; McAlonan, H.; Montgomery, D.; Stevenson, P. J. J. Chem. Soc., Perkin
Trans. 1 2000, 1129-1137.
8.
Hickman, V.; Kondoh, A.; Gabor, B.; Alcarazo, M.; Fürstner, A. J. Am. Chem. Soc. 2011,
133, 13471-13480.
9.
Min, G. K.; Skrydstrup, T. J. Org. Chem. 2012, 77, 5894-5906.
10.
Prugh, J. D.; Birchenough, L. A.; Egbertson, M. S. Synth. Commun. 1992, 16, 2357-2360
11.
Neitzel, M.; Zinner, G. Liebigs Ann. Chem. 1980, 11, 1907-1912.
12.
Shendage, D. M.; Fröhlich, R.; Haufe, G. Org. Lett. 2004, 6, 3675-3678.