OPTIMIZATION OF SCALE-UP SYNTHESIS OF ANTI-CANCER CERAMIDE ANALOG 315

Abstract

Ceramides, serve as central mediators in sphingolipid metabolism and signaling pathways. They function in signaling events which induce apoptosis, cell cycle arrest, and autophagic responses. In cancer cells, ceramide levels are often suppressed by the up-regulation of ceramide-metabolizing enzymes or the down-regulation of ceramide-generating enzymes, resulting in increased cancer cell survival. Chemotherapeutic drugs and radiation therapy have been shown to increase intracellular ceramide levels leading to anti-cancer effects. Anti-cancer effects have also been seen in cancer cells with the use of exogenous short-chain ceramides. Our laboratory has synthesized a library of ceramide analogs and tested their effects on breast cancer cell lines. Analog 315 has been shown to be the most effective ceramide analog in our library. Here, we are reporting a large-scale synthesis of that analog is reported.

Introduction

Ceramides, also known as “tumor suppressor lipids,” serve as central mediators in sphingolipid metabolism and signaling pathways (1–3). They function in signaling events to induce apoptosis, cell cycle arrest, and autophagic responses (3). In typical cancerous environments, ceramide levels are often suppressed by the up-regulation of ceramide-metabolizing enzymes or the down-regulation of ceramide-generating enzymes, resulting in the enhancement of cancer cell proliferation, migration, and survival (3–5). Chemotherapeutic drugs and radiation therapy have shown to increase intracellular ceramide levels leading to the observed anti-cancer effects (2). Anti-cancer effects have also been seen in cancer cells with the use of short-chain ceramides (2). Introduction of synthetic ceramide analogs to cancer cells has the potential to inhibit ceramide-metabolizing enzymes, essentially regulating ceramide levels necessary for promotion of apoptosis (6). Ceramide analogs have also been shown to promote apoptosis by interacting with ceramide down-stream targets. In addition, previous studies have shown that ceramides show preferred inhibition on the growth of multi-drug resistant cancer cells in comparison to drug-sensitive cancer cells (7–10). The up-regulation of ceramide levels and associated activities seems to be a viable potential strategy for cancer treatment.

A large library of ceramide analogs have been previously synthesized and reported.(12–14). These analogs have been tested in clonogenic survival and pro-apoptotic assays in chemo-sensitive MCF-7, endocrine-resistant MDA-MB-231, and chemo-resistant MCF-7TN-R breast cancer cell lines. The latter two cell lines represent highly aggressive, metastatic, and drug-resistant forms of human breast cancer. MDA-MB-231 cell lines are triple-negative (ER-R/PR/HER2 negative) and resistant to hormone and endocrine therapies. MCF-7TN-R cell lines were derived from MCF-7 cells grown in increasing concentrations of tumor necrosis factor until resistance was established (11). These cells exhibit increased resistance to several chemotherapeutic agents including etoposide, paclitaxel, and doxorubicin leading to treatment failure and higher mortality rates in patients. In all cell lines tested, we found Analog 315 was found to be the most effective ceramide analog in inducing apoptosis (14).

This analog also shows higher activity in chemo-resistant breast cancer cells. To perform further in vivo studies, bulk quantities of Analog 315 were required, and synthesized by a scaled-up procedure discussed here.

Experimental

Synthesis of Analog 315 was previously reported, and the scheme of the synthesis is shown in Figure 1 (12–14). In the original synthetic scheme, boc-serine [1] (Aldrich, USA) was coupled with amine [2] (1.1 equivalents, tetradecyl amine, TCI, Japan) to get dipeptide [3]. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, 1.1 equivalents, Bachem, Germany) and 1-hydroxy benzotriazole (HOBt, 1.1 equivalents, Aldrich, USA) were used as coupling reagents. 4-Methyl-morpholine (3.0 equivalents, Sigma, USA) was used as a base. The boc (tert-buty ether) group was then cleaved using trifluoroacetic acid (TFA, 2.5 equivalent, Alfa Aesar, USA) to give the free amine [4], which was further reacted with salicaldehyde (1.0 equivalent Acros Organic, China) in the presence of strong base, sodium hydroxide (3.0 equivalents, Sigma Aldrich, USA), to get Analog 315 [5].

Figure 1.

The originally reported synthetic scheme for synthesis of Analog 315 (12–14).

While scaling up the reaction, it was noticed that modifications were only needed in reaction step 1 where large amounts of side product urea were generated. To modify step 1, a different coupling reagent, N,N′-dicyclohexyl-carbodiimide (DCC, 1.1 equivalence, Sigma Aldrich, USA), was used to replace EDCI, in addition to a different base diisopropyl ethyl amine (DIPEA, AIC scientific, USA) (Table 1).

Table 1.

Different reaction conditions for reaction step 1.

Trail No.	Reagent for 1St step	Base used	Yield (%)
1	DCC/HOBt	N-methyl-morpholine	89
2	EDCI/HOBt	N-methyl-morpholine	90
3	EDCI/HOBt	Diisopropyl ethyl amine	90.7

The above reaction was performed on 1.0 g, 5.0 g, and 10.0 g scales, and the product was recovered and used without any further purification. Once the dipeptide was obtained, the boc (tert-butyl carbamate) group was cleaved using trifluoroacetic acid (2 equivalents) in dichloromethane as a solvent. The product was concentrated using rotary evaporator, further dried under high vacuum overnight, and used for the final step without any further purification. The amine-TFA [4] (5.0 g, 12.58 mmol, 1.0 equivalent) was coupled with salicaldehyde (1.5 mL, 13.84 mmol, 1.1 equivalents) and sodium hydroxide as base (1.0 g, 25.58 mmol, 2.0 equivalents) in methanol (50 mL) for 6 hours to give the final product. The reaction mixture was filtered, the crude product was concentrated on rotary evaporator, and purified using column chromatography (silica gel, 60–120 Mesh, 5% MeOH: 95% dichloromethane). The final step for the synthesis of Analog 315 was performed on a 5.0g scale.

GC/MS (m/e): 403 [M_H]⁺, 387 [M-OH]^{+. 1}H NMR (DMSO-d6, 300 MHz) δ/ppm = 8.5 (s, 1H), 7.9 (t, J = 5.5 Hz, 1H), 7.5 (d, J = 7.5 Hz, 1H), 7.3 (t, J = 7.5 Hz, 1H), 6.9 (m, 2H), 4.9 (br, 1H), 3.9 (dd, J = 4.7 Hz, J = 7.8 Hz, 1H), 3.8 (dd, J = 4.5 Hz, J = 10.8 Hz, 1H), 3.6 (m, 1H), 3.1 (m, 2H), 1.4 (m, 2H), 1.2 (m, 22H), and 0.8 (t, J = 6.0 Hz, 3H). ¹³C NMR (DMSO-d6, 75 MHz) δ/ppm = 169.571, 167.278, 161.022, 133.115, 132.478, 119.587, 119.238, 117.127, 75.115, 63.408, 39.176, 31.979, 29.717, 29.656, 29.398, 26.983, 22.778, and 14.624.

Results and Discussion

For the original coupling reaction, a yield of 89% was achieved using DCC. Changing EDCI with DCC did not increase the yield drastically; however, it simplified the work-up of the crude product. DCC is a well-known dehydrating agent, however it was noticed that during the workup some urea was left in the organic layer. This problem was overcome by using EDCI. Changing the base did not lead to a major difference in the reaction yield (Table 1).

Conclusion

To conclude, the synthesis of Analog 315 was achieved on a large scale. While doing this, step 1 was modified using a different coupling reagent as well as a different base. Steps 2 and step 3 were also performed on a large scale.

Figure 2.

Updated synthesis scheme of Analog 315.