molecules Article
Synthesis, Experimental and Density Functional Theory (DFT) Studies on Solubility of Camptothecin Derivatives Chin-Hung Lai 1,2, * , Chia-Chin Chang 3 , Yi-Lin Weng 3 and Ta-Hsien Chuang 3,4, * 1 2 3 4
*
Department of Medical Applied Chemistry, Chung Shan Medical University, Taichung 40201, Taiwan Department of Medical Education, Chung Shan Medical University Hospital, Taichung 40201, Taiwan Master Program for Pharmaceutical Manufacture, China Medical University, Taichung 40402, Taiwan;
[email protected] (C.-C.C.);
[email protected] (Y.-L.W.) School of Pharmacy, China Medical University, Taichung 40402, Taiwan Correspondence:
[email protected] (C.-H.L.);
[email protected] (T.-H.C.); Tel.: +886-4-2473-0022 (ext. 12224) (C.-H.L.); +886-4-2205-3366 (ext. 5150) (T.-H.C.); Fax: +886-4232-4818 (C.-H.L.); +886-4-2207-8083 (T.-H.C.)
Received: 7 November 2018; Accepted: 30 November 2018; Published: 1 December 2018
Abstract: Two camptothecin derivatives, 10-cyclohexyl-7-methyl-20(S)-camptothecin and 7-methyl10-morpholino-20(S)-camptothecin, were synthesized and their differences in solubility were investigated using four chosen solvent systems. Based on our results, 10-cyclohexyl-7-methyl20(S)-camptothecin exhibited higher solubilities than 7-methyl-10-morpholino-20(S)-camptothecin in polar aprotic solvents. However, these two camptothecin derivatives did not exhibit apparent differences in solubility between 5% dimethyl sulfoxide (DMSO)/95% normal saline co-solvent system and 5% dimethylacetamide (DMAC)/95% normal saline co-solvent system. To rationalize their differences in solubility, we also tried to perform a DFT-B3LYP study to investigate their interaction with one water molecule. Keywords: acid-catalyzed; condensation; camptothecin; solubility; DFT
1. Introduction Camptothecin (CPT, 1, Figure 1) is a pentacyclic alkaloid first isolated from Camptotheca acuminata by Wall and coworkers in the early 1960s [1]. In the late 1980s, DNA topoisomerase I (topo I) was found to be the therapeutic target for CPT, putting it on the frontline of anticancer drug development [2–5]. Therefore, its total synthesis, mechanism of action, structure-activity relationship (SAR), and pharmacology have been studied extensively, and preclinical studies and clinical trials of CPT have been carried out. As a result of these research efforts, three CPT analogs, topotecan (TPT, 2), irinotecan (CPT-11, 3), and belotecan (CKD-602, 4) have been used for the clinical treatment of ovarian, small-cell lung, and refractory colorectal cancers [6–8]. Their clinical success and intriguing mechanism of action have stimulated great interest in further exploration of CPT derivatives with better antitumor activity; several such derivatives are now undergoing preclinical evaluation. With the three successful compounds 2–4 in clinical practice and 10 additional compounds 5–14 in clinical trials (Figure 2), CPT analogs have become highly relevant clinical anticancer compounds. Moreover, some excellent reviews on CPT derivatives have been published [9–20].
Molecules 2018, 23, 3170; doi:10.3390/molecules23123170
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2 2ofof1111 9 10 9
10 11 11
7
8
A
8
A
13
N B
2
13
N
2
12 12
5
6
7 B
6
O
C5 N O D 3 N C E O D 3 E O OH O
CPT (1)
(1) OH O1. FigureCPT 1. Structure of CPT, Figure 1. Structure Structure of of CPT, CPT, 1.
HN N
N
HO N
N
O N
HO N
HN
O
N
O
N O
O
O N N
O
O
N
N
N
N
O
O
N O
N
Topotecan (2)
O OH O
Irinotecan (3)
O OH O
Belotecan (4)
O OH O
Topotecan (2)
OH O
Irinotecan (3)
OH O
Belotecan (4) O
OH O
N
NH2 NH2 N F
O
N
N
F
O N
N
N
O
O
O
O O
N
O
N
N O
N Exatecan (5)
O OH O
Exatecan (5) R
OH O
R
N
N
O O
HO2C
O
O
O
O O O
O N N
Lurtotecan (6)
O N Sinotecan (7)
Lurtotecan (6)
OH O
Sinotecan (7)
N Gimatecan (10)
9-NC (9): R= NH2
Gimatecan (10)
2
N
O
N
N O
N
OH O Rubitecan (8): R= NO 2 9-NC (9): R= NH 2 Rubitecan (8): R= NO
O
O N
O OH O
OH O
Si
N O
O O OH O
Si
O N O
O
O
N
O OH O
N
N
HO2C O
N
O N
N
O
N O
N
O OH O OH O
Karenitecin (11)
O OH O
Karenitecin (11)
OH O
Si N
Si HO N
HO N
O
F
O
F F
N
F
N
N O
N
O OH O
N
O N
O N
O
N O O OH O
Cl
N
Cl
N
O
N O
Elomotecan (14)
O OH O
DB-67 (12) Diflomotecan (13) (14) O O practice Elomotecan clinical or clinical trials. Figure 2. OH Structures of CPT analogues in use inOH
OH O
DB-67 (12)
Diflomotecan (13)
Figure 2. Structures of CPT analogues in use in clinical practice or clinical trials.
Despite due to Despite its its impressive impressive activity activity against against various various cancers cancers in in experimental experimental systems, systems, due to its its negligible water solubility, CPT was not recognized as a frontline anticancer drug until its mechanism negligible water was not recognized as acancers frontline drugsystems, until its mechanism Despite its solubility, impressiveCPT activity against various inanticancer experimental due to its of action was was elucidated elucidatedininthe the1980s. 1980s.In In early 1970s, the water-soluble sodium salt of of action thethe early 1970s, onlyonly the water-soluble sodium salt of CPT negligible water solubility, CPT was not recognized as a frontline anticancer drug until its mechanism CPT (15, Figure 3) had been tested in a clinical trial [21]. However, 15 has low efficacy and causes (15,action Figure had been in tested in a clinical trial 1970s, [21]. However, 15 has low efficacy causes of was3)elucidated the 1980s. In the early only the water-soluble sodium and salt of CPT unpredictable and severe side effects, including hemorrhagic cystitis and myelotoxicity. Therefore, unpredictable side effects, including cystitis Therefore, (15, Figure 3) and had severe been tested in a clinical trialhemorrhagic [21]. However, 15 and has myelotoxicity. low efficacy and causes unpredictable and severe side effects, including hemorrhagic cystitis and myelotoxicity. Therefore,
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clinical trials of CPT were discontinued for more than a decade [22,23]. Thus, the water solubility of CPT has had a great impact on its clinical application. O N
OH
N
ONa 15
OH O
Figure 3. The sodium salt of CPT 15.
Water solubility physical and and chemical chemical property property of of organic organic small small molecule molecule drugs drugs and and solubility is Water is aa virtual virtual physical Drugs with with better better water water solubility solubility often often exhibit exhibit higher higher potency potency a very important issue in drug design. Drugs favorable pharmacokinetic than drugs with lower water solubility. Structural and more profiles with lower water solubility. more favorable pharmacokinetic than drugs Structural modification is is aa straightforward and effective effective method method to to enhance enhance water water solubility. solubility. One One method method of of straightforward and modification The introduction of of polar groups cancan increase the structural modification modificationisistotointroduce introducea apolar polargroup. group. The introduction polar groups increase hydration of theofcompounds and promote the thermodynamic process of dissolution. For example, For the the hydration the compounds and promote the thermodynamic process of dissolution. introduction of a morpholine ring at the 6 position of the nitrogen-containing parent ring of rilpivirine example, the introduction of a morpholine ring at the 6 position of the nitrogen-containing parent (16, 4) increased its water solubility from onlysolubility 0.02 µg/mL to 27.3 [24]. Rilpivirine is a ringFigure of rilpivirine (16, Figure 4) increased its water from onlyµg/mL 0.02 μg/mL to 27.3 μg/mL non-nucleoside reverse transcriptase inhibitor that has good activity against wild-type HIV-1 and two [24]. Rilpivirine is a non-nucleoside reverse transcriptase inhibitor that has good activity against wildmajorHIV-1 mutants. The major introduction of The the polar morpholine increased the EC rilpivirine type and two mutants. introduction of thering polar morpholine ring increased the from EC 50 50 of 0.00067 to 0.0086 wild-type HIV-1. of rilpivirine fromµmol/L 0.00067for to 0.0086 μmol/L for wild-type HIV-1. CN
CH3
HN
HN N
N
N H Rilpivirine (16)
CN
CN
N
O N
O
N
N H
Figure 4. of of rilpivirine (16)(16) withwith a polar morpholine moiety to improve its water 4. Functionalization Functionalization rilpivirine a polar morpholine moiety to improve its solubility. water solubility.
analogues have have been been substituted substituted at at the the 7, 7, 9, 9, 10, 10, or or 11 11 positions. positions. As shown in Figure 2, CPT analogues Numerous SAR studies have shown that 7-, 9-, and 10-substituted CPT analogs are better tolerated or SAR studies have shown that 7-, 9-, and 10-substituted CPT analogs are better tolerated have higher anticancer activity than than the parent compound, CPT. For example, the hydrolysis product or have higher anticancer activity the parent compound, CPT. For example, the hydrolysis and activeand metabolite of irinotecinof (3),irinotecin 7-ethyl-10-hydroxycamptothecin, in which the carbamate group product active metabolite (3), 7-ethyl-10-hydroxycamptothecin, in which the has been removed andbeen an OH group isand present at position could at possibly be 10, important in terms be of carbamate group has removed an OH group is10, present position could possibly either increasing theofwater solubility or decreasing the unwanted stabilization the open stabilization hydroxyacid important in terms either increasing the water solubility or decreasing theof unwanted form human albumin.form Moreover, a recent x-rayMoreover, crystallographic of the ternary complex of theby open hydroxyacid by human albumin. a recentanalysis x-ray crystallographic analysis formed by topo I, a DNA oligonucleotide 2 showed that substituents at thethat 7 and 9 positions of the ternary complex formed by topo I, a and DNA oligonucleotide and 2 showed substituents at of CPT did not influence thedid CPT-topo I interactions [25]. The 7–10 positions can 7–10 bear large groups, the 7 and 9 positions of CPT not influence the CPT-topo I interactions [25]. The positions can allowing wide variety of structural modifications. In addition to substitution at positions 7–10, fusion bear largea groups, allowing a wide variety of structural modifications. In addition to substitution at of an additional ring onto theadditional A/B ring has to several clinicalCPT trialsanalogues (as depicted positions 7–10, fusion of an ringled onto the A/BCPT ringanalogues has led toinseveral in in Figure 2). (as In this study, 10 position was chosen forposition substitution with either a cyclohexyl or clinical trials depicted inthe Figure 2). In this study, the 10 was chosen for substitution with morpholine ring (depicted in Figure 5) to investigate the effect ofinvestigate the water solubility on its either a cyclohexyl or morpholine ring (depicted in Figure 5) to the effect of ofCPT the water anticancerofactivity. discussed, introduction of a morpholine group of should increase solubility CPT onAs itspreviously anticancer activity. Asthe previously discussed, the introduction a morpholine the water solubility of CPT,the andwater thus influence potency group should increase solubilityits of CPT, and andpharmacokinetic thus influenceprofile. its potency and pharmacokinetic profile.
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O O N N
O O
N N
N N
N N
O O
N N O O
P210 P210
O O P211 P211
O OH O OH
O OH O OH
Figure 5. 5. The The CPT CPT analogues analogues discussed discussed in in this this study. study. Figure The CPT analogues discussed
2. Results and Discussion 2. Results Results and and Discussion Discussion 2. 2.1. Synthesis of CPT Derivatives P210 and P211 2.1. Synthesis Synthesis of of CPT CPT Derivatives Derivatives P210 P210 and and P211 P211 2.1. The strategy for the preparation preparation of of P210 P210 and and P211 P211 isisshown shownininScheme Scheme1.1.The The condensation The strategy strategy for for the the condensation of The preparation of P210 and P211 is shown in Scheme 1. The condensation of of commercially available 1-(2-amino-5-morpholinophenyl)-1-ethanone (16a) with (S)-4-ethyl-4commercially available 1-(2-amino-5-morpholinophenyl)-1-ethanone (16a) with (S)-4-ethyl-4commercially available 1-(2-amino-5-morpholinophenyl)-1-ethanone (16a) with (S)-4-ethyl-4hydroxy-7-8-dihydro-1H-pyrano[3,4-f ]indolizine-3,6-10(4H)-trione (17) (17) in in the presence of hydroxy-7-8-dihydro-1H-pyrano[3,4-f]indolizine-3,6-10(4H)-trione the presence presence of of aaa catalytic catalytic hydroxy-7-8-dihydro-1H-pyrano[3,4-f]indolizine-3,6-10(4H)-trione (17) in the catalytic amount of p-toluenesulfonic acid monohydrate (p-TSA) in toluene under reflux for 3 h produced a lowa amount of of p-toluenesulfonic p-toluenesulfonic acid acid monohydrate monohydrate (p-TSA) (p-TSA) in in toluene toluene under under reflux reflux for for 33 hh produced produced amount a yield of P211 [26]. The p-TSA catalyzed condensation could be carried out in a sealed tube by heating low yield of P211 [26]. The p-TSA catalyzed condensation could be carried out in a sealed tube by low yield of P211 [26]. The p-TSA catalyzed condensation could be carried out in a sealed tube by at 130 ◦ C overnight, affording P211 inaffording 83% yield. Moreover, heating at 130 130 °C °C overnight, P211 in in 1-(2-amino-5-cyclohexylphenyl)ethanone 83% yield. yield. Moreover, Moreover, 1-(2-amino-51-(2-amino-5heating at overnight, affording P211 83% (16b) was synthesized by the acylation of 4-cyclohexylamine [27].ofThen, P210 could be[27]. obtained cyclohexylphenyl)ethanone (16b) was synthesized by the acylation 4-cyclohexylamine Then, cyclohexylphenyl)ethanone (16b) was synthesized by the acylation of 4-cyclohexylamine [27]. Then, at an could 80% yield by the p-TSA catalyzed condensation of 16b with 17 under the above-mentioned P210 be obtained at an 80% yield by the p-TSA catalyzed condensation of 16b with 17 under P210 could be obtained at an 80% yield by the p-TSA catalyzed condensation of 16b with 17 under optimized conditions. optimized conditions. the above-mentioned above-mentioned the optimized conditions. 1) AlCl AlCl3,, BCl BCl3 1) 3 3 CH CN, toluene 3 CH3CN, toluene reflux, 22 hh reflux,
RR
O O
2) 1MHCl, 1MHCl, 80 80 ° ° C, 33 hh NH2 2) C, NH 2
NH2 NH 2 16a, RR == morpholino morpholino 16a, 16b, RR == cyclohexyl cyclohexyl (73 (73 %) %) 16b, ++ NN
O O
cat. cat. RR p-TSA p-TSA toluene toluene sealed tube tube sealed 130 ° ° 130 CC overnight overnight
O O O O OH O O OH
NN
O O
NN O O
O OH O OH P211, RR == morpholino morpholino (83 (83 %) %) P211, P210, R = cyclohexyl (80 %) P210, R = cyclohexyl (80 %)
17 17
Scheme 1. Preparation of P210 and P211. Scheme1. 1. Preparation Preparation of of P210 P210 and andP211. P211. Scheme
2.2. The Difference in Solubility between P210 and P211 2.2. The The Difference Difference in in Solubility Solubility between between P210 P210 and and P211 P211 2.2. The solubilities of P210 and P211 in DMSO, DMAC, 5% DMSO/95% normal saline co-solvent The solubilities solubilities of of P210 P210 and and P211 P211 in in DMSO, DMSO, DMAC, DMAC, 5% 5% DMSO/95% DMSO/95% normal normal saline saline co-solvent co-solvent The system, and 5% DMAC/95% normal saline co-solvent system were summarized in Table 1. system, and 5% DMAC/95% normal saline co-solvent system were summarized in Table 1. 1010system, and 5% DMAC/95% normal saline co-solvent system were summarized in Table 1. 10-Cyclohexyl-7-methyl-20(S)-camptothecin (P210) exhibited higher solubilities than 7-methyl-10Cyclohexyl-7-methyl-20(S)-camptothecin (P210) (P210) exhibited exhibited higher higher solubilities solubilities than than 7-methyl-107-methyl-10Cyclohexyl-7-methyl-20(S)-camptothecin morpholino-20(S)-camptothecin (P211) in polar aprotic solvents (DMSO and DMAC). However, these morpholino-20(S)-camptothecin (P211) in polar aprotic solvents (DMSO and DMAC). However, morpholino-20(S)-camptothecin (P211) in polar aprotic solvents (DMSO and DMAC). However, two camptothecin derivatives P210 and P211 did not exhibit apparent differences in solubility between these two camptothecin derivatives P210 and P211 did not exhibit apparent differences in solubility these two camptothecin derivatives P210 and P211 did not exhibit apparent differences in solubility 5% DMSO/95% normal saline co-solvent system and 5% DMAC/95% normal saline co-solvent system. between 5% 5% DMSO/95% DMSO/95% normal normal saline saline co-solvent co-solvent system system and and 5% 5% DMAC/95% DMAC/95% normal normal saline saline cocobetween solvent system. solvent system.
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Table 1. The solubilities of P210 and P211 (in mg/1 mL). Table 1. The solubilities of P210 and P211 (in mg/1 mL).
P210 10.6 P210 DMSO sparingly soluble 10.6 DMSO 49.5 sparingly soluble DMAC soluble49.5 DMAC 0.10soluble 5% DMSO + 95% normal saline 0.10 5% DMSO + 95% normal salinevery slightly soluble very slightly soluble 0.10 0.10 5% DMAC + 95%+normal saline 5% DMAC 95% normal saline very slightly soluble very slightly soluble
P211 7.2 P211 slightly soluble 7.2 13.2 slightly soluble 13.2soluble sparingly sparingly 0.10soluble 0.10 very slightly soluble very slightly soluble 0.10 0.10 very soluble very slightly slightly soluble
2.3. 2.3.DFT-B3LYP DFT-B3LYP Study Study of of P210 P210(P211) (P211)and andTheir TheirHydrated HydratedComplexes Complexes The ring should increase the the polarity of CPT, and and thus,thus, the The introduction introductionofofthe themorpholine morpholine ring should increase polarity of CPT, solubility of CPT in a polar solvent, e.g. water, our solvent of interest. However, the results of the the solubility of CPT in a polar solvent, e.g. water, our solvent of interest. However, the results aforementioned solubility test were different. Therefore, we attempted to rationalize the differences of the aforementioned solubility test were different. Therefore, we attempted to rationalize the in solubility in from a theoretical We could calculate the dipole moments of both P210 and differences solubility from aspect. a theoretical aspect. We could calculate the dipole moments ofP211 both using the B3LYP/D95V++** theoretical level to perform a gas-phase study. Generally, a more polar P210 and P211 using the B3LYP/D95V++** theoretical level to perform a gas-phase study. Generally, molecule has amolecule larger dipole and better solubility in a polar solvent a less polarthan molecule. a more polar has amoment larger dipole moment and better solubility in than a polar solvent a less The dipole moments of P210 and P211 are listed and compared in Figure 6, which shows that the polar molecule. The dipole moments of P210 and P211 are listed and compared in Figure 6, which introduction of a polar morpholine ring in position 10 of CPT slightly reduced its dipole moment and shows that the introduction of a polar morpholine ring in position 10 of CPT slightly reduced its dipole consequently polarity. its polarity. moment and its consequently
Figure 6. Compounds P210 and P211 (the values in parentheses are their respective dipole moments in Figure Compounds and P211 (theoxygen valuesininred, parentheses are their respective dipole moments Debye;6.carbon in gray,P210 nitrogen in blue, and hydrogen in white). in Debye; carbon in gray, nitrogen in blue, oxygen in red, and hydrogen in white).
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We not only compared the differences in their dipole moments, but also compared the differences thecompared strength of interaction with onemoments, water molecule. A CPT derivative with We notinonly thetheir differences in their dipole but also compared the differences stronger interaction with one water molecule should have better aqueous solubility than that with in the strength of their interaction with one water molecule. A CPT derivative with stronger interaction weaker interaction with one water molecule. In agreement with previous studies [28,29], both P210 with one water molecule should have better aqueous solubility than that with weaker interaction with and P211 interact most strongly with the water molecule via the carbonyl group in the D ring (Figure one water molecule. In agreement with previous studies [28,29], both P210 and P211 interact most 7). strongly with the water molecule via the carbonyl group in the D ring (Figure 7).
0.9816 (0.9673)
1.823
1.253 (1.244)
P210
0.9818 (0.9673)
1.821
1.254 (1.244)
P211 Figure 7. Cont.
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Figure 7. Cont. Molecules 2018, 23, 3170
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0.9863 (0.9673) 1.939
P211
0.9767 (0.9673) 1.881
P211 Figure 7. 7. Hydrated HydratedP210 P210and and P211. P211. Values Valueswithout withoutparentheses parenthesesindicate indicatebond bondlengths lengths(Å (Å) for the the Figure ) for water/CPTderivative derivativesystem; system;values valuesininparentheses parenthesesindicate indicatebond bondlengths lengths(Å(Å) forthe thefree freemolecules molecules water/CPT ) for (carbonin ingray, gray, nitrogen nitrogen in in blue, blue, oxygen oxygen in in red, red, and and hydrogen (carbon hydrogen in in white). white) .
Asdepicted depictedin inFigure Figure 7, 7, the the C=O C=Obond bondlength lengthincreases increasesin inboth bothP210 P210and andP211 P211upon uponinteraction interaction As with a water molecule. At this theoretical level, without correcting for the basis set superposition error, with a water molecule. At this theoretical level, without correcting for the basis set superposition the strengths of theof interactions of P210 and P211 with awith single waterwater molecule werewere calculated to be error, the strengths the interactions of P210 and P211 a single molecule calculated − 25.2 and − 28.3 kJ/mol, respectively. The basis set superposition error is not corrected in this study to be −25.2 and −28.3 kJ/mol, respectively. The basis set superposition error is not corrected in this because this correction must be practically equal for all the and cannot change change the relative study because this correction must be practically equal for systems all the systems and cannot the order of individual hydration energies. Apart from the carbonyl group on ring D, both P210 and P211 relative order of individual hydration energies. Apart from the carbonyl group on ring D, both P210 haveP211 other possible sites: the sites: oxygen on ring both C=O and C=O OH group on group ring E, and have other hydration possible hydration theatom oxygen atomE,on ring E, both and OH and theE,nitrogen on ring interaction energiesenergies of these of sites forsites P210for (P211) a water on ring and the atom nitrogen atomB;onthe ring B; the interaction these P210with (P211) with molecule were calculated to be − 13.7 ( − 16.8), − 15.7 ( − 18.0), and − 8.3 ( − 11.5) kJ/mol, respectively. a water molecule were calculated to be −13.7 (−16.8), −15.7 (−18.0), and −8.3 (−11.5) kJ/mol, respectively. Interestingly, the the C=O C=Oand andOH OHgroups groupson onring ringEEfor for both both P210 P210 and and P211 P211 could could simultaneously simultaneouslyform form Interestingly, O . . . H hydrogen bonds with one water molecule. Such interactions were still weaker than that
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between the carbonyl group on ring D and a water molecule. It was worthwhile to mention that the nitrogen and oxygen atoms on the morpholine ring in P211 can also form a hydrogen bond with one water molecule; the corresponding hydrogen bond complex is also depicted in Figure 7. Using the same theoretical method, we found that the hydrogen bond strengths with one water molecule were −4.1 and −10.9 kJ/mol for nitrogen and oxygen, respectively. This could be easily rationalized by the fact that oxygen is more electronegative than nitrogen. Although the calculated hydrogen bond lengths of P210 and P211 with one water molecule did not show any significant difference, P211 could form a stronger interaction with one water molecule. Based on our recently calculated results, although a morpholine ring is a polar group, its introduction into CPT reduced the dipole moment of CPT and did not induce a large difference in the strength of the interaction between CPT and a water molecule. Therefore, P211 might not have better aqueous solubility than P210. 3. Materials and Methods 3.1. General Information Melting points (mp) were determined using a Büchi M560 apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and are uncorrected. Optical rotations ([α]D ) were measured using a P-2000 polarimeter (JASCO, Easton, MD, USA). Infrared (IR) spectra were reported in wave numbers (cm−1 ) using an IR Prestige-21/FTIR-8400S Fourier transform infrared spectrophotometer (Shimadzu Kyoto, Kyoto Prefecture, Japan). 1 H and 13 C-NMR spectra were recorded using a Bruker AVANCE III 500 FT-NMR spectrometer (Bruker, Rheinstetten, Germany) at room temperature, and the chemical shifts are reported in ppm using tetramethylsilane (TMS) as the internal standard. Mass spectra (MS) were obtained using a LCQ ion-trap mass spectrometer using the electrospray ionization (ESI) technique (Thermo-Finnigan, Mundelein, IL, USA). High-resolution electrospray ionization mass spectra (HRESI-MS) were recorded on a LTQ orbitrap XL hybrid ion trap-orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA). Column chromatography was performed on 230−400 mesh silica gel. 3.2. Synthesis of 1-(2-Amino-5-cyclohexylphenyl)ethanone (8) To a slurried mixture of 4-cyclohexylaniline (175.3 mg, 1.0 mmol) and anhydrous aluminum chloride (186.7 mg, 1.4 mmol) in dry toluene (6 mL) was added boron trichloride (1 M in hexane, 6.9 mL, 1.2 mmol) at room temperature under N2 . Acetonitrile (2.6 mL, 50 mmol) was added to the reaction mixture, and the mixture was refluxed under N2 for 3 h. After cooling, 1M aqueous hydrochloric acid (2 mL) was added to the resulting mixture at room temperature. The mixture was stirred at 80 ◦ C for 0.5 h, and then poured into ice water. A 5 M aqueous sodium hydroxide solution was added to increase the pH of the resultant solution to >13, and the mixture was extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (10 mL × 1), dried over MgSO4 , filtered, and concentrated. The crude product was purified by column chromatography using silica gel and hexane−EtOAc (20:1 v/v) as the eluent, affording the pure product 8 as an off-white solid (158.6 mg, 73% yield). mp 69–69.5 ◦ C; IR (KBr) νmax 3429, 3329, 2924, 2846, 1643, 1632, 1547 cm−1 ; 1 H-NMR (500 MHz, CDCl ) δ 1.17–1.44 (5H, m), 1.70–1.90 (5H, m), 2.35–2.45 (1H, m), 2.60 (3H, s), 6.12 3 (2H, br s), 6.59 (1H, d, J = 8.4 Hz), 7.14 (1H, d, J = 8.4 Hz), 7.50 (1H, s); 13 C-NMR (125 MHz, CDCl3 ) δ 26.0, 26.8 (C × 2), 27.8, 34.6 (C × 2), 43.5, 117.2, 118.1, 129.4, 133.3, 135.4, 148.4, 200.7; ESIMS m/z (rel. int.) 218 (100, [M + H]+ ); HRESIMS m/z calcd for C14 H20 NO 218.1539; found 218.1541 [M + H]+ . 3.3. General Procedure for the Condensation of 16 with 17 A mixture of compound 16a or 16b (0.1 mmol), (S)-4-ethyl-4-hydroxy-7-8-dihydro-1H-pyrano[3,4-f ]indolizine-3,6-10(4H)-trione (17, 0.2 mmol), and p-TSA (0.02 mmol) in dry toluene (6 mL) was placed in a sealed tube and then heated at 130 ◦ C overnight. After cooling, the resulting solution
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was directly purified by column chromatography using silica gel and pure CHCl3 or CH2 Cl2 -MeOH (40:1 v/v) as the eluent, affording the pure camptothecin derivative P210 or P211. 10-Cyclohexyl-7-methyl-20(S)-camptothecin (P210). Eluted with pure CHCl3 ; yield 80%; pale yellow solid; mp 243–245 ◦ C; [α]D +37.3◦ (c 0.32, CHCl3 ); IR (KBr) νmax 3433, 3337, 2924, 2851, 1748, 1659, 1597 cm−1 ; 1 H-NMR (500 MHz, CDCl3 ) δ 1.03 (3H, t, J = 7.4 Hz), 1.29–1.38 (1H, m), 1.42–1.62 (4H, m), 1.80–1.94 (5H, m), 1.99–2.02 (2H, m), 2.73–2.80 (1H, m), 2.76 (3H, s), 3.98 (1H, s), 5.21 (2H, s), 5.29 (1H, d, J = 16.2 Hz), 5.73 (1H, d, J = 16.2 Hz), 7.65 (1H, s), 7.69 (1H, d, J = 8.7 Hz), 7.82 (1H, s), 8.14 (1H, d, J = 8.7 Hz); 13 C-NMR (125 MHz, CDCl3 ) δ 7.8, 15.3, 26.1, 26.8 (C × 2), 31.7, 34.4 (C × 2), 45.0, 49.8, 66.4, 72.8, 97.8, 118.2, 120.2, 127.6, 128.0, 130.2, 130.3, 139.1, 147.3, 147.8, 147.9, 150.2, 150.8, 157.7, 173.9; ESIMS m/z (rel. int.) 445 (100, [M + H]+ ), 401 (24), 381 (30); HRESIMS m/z calcd for C27 H29 N2 O4 445.2122; found 445.2123 [M + H]+ . 7-Methyl-10-morpholino-20(S)-camptothecin (P211). Eluted with CH2 Cl2 -MeOH (40:1 v/v); yield 83%; yellow solid; mp 283–285 ◦ C; [α]D +11.3◦ (c 0.34, CHCl3 ); IR (KBr) νmax 3414, 1744, 1655, 1593 cm−1 ; 1 H-NMR (500 MHz, CDCl ) δ 1.03 (3H, t, J = 7.2 Hz), 1.80–1.98 (2H, m), 2.70 (3H, s), 3.38 (4H, t, 3 J = 4.8 Hz), 3.85 (1H, s), 3.95 (4H, t, J = 4.8 Hz), 5.19 (2H, s), 5.30 (1H, d, J = 16.1 Hz), 5.74 (1H, d, J = 16.1 Hz), 7.14 (1H, s), 7.55 (1H, d, J = 9.3 Hz), 7.58 (1H, s), 8.10 (1H, d, J = 9.3 Hz); 13 C-NMR (125 MHz, CDCl3) δ 7.8, 15.4, 31.6, 48.8 (C × 2), 49.8, 66.4, 66.7 (C × 2), 72.9, 97.2, 104.2, 117.5, 122.0, 128.2, 129.3, 131.3, 137.2, 144.3, 147.5, 148.8, 150.1, 150.2, 157.7, 174.0; ESIMS m/z (rel. int.) 448 (100, [M + H]+ ), 404 (13), 381 (79); HRESIMS m/z calcd for C25 H26 N3 O5 448.1867; found 448.1871 [M + H]+ . 3.4. The Solubility Test To test the difference in solubility between P210 and P211, four solvents were chosen in this study for a systematic comparison; namely, DMSO, DMAC, 5% DMSO/95% normal saline (v/v), and 5% DMAC/95% normal saline (v/v). 3.5. DFT-B3LYP Study of P210 (P211) and Their Hydrated Complexes All DFT calculations were carried out by the program Gaussian 16 (Wallingford, CT, USA) [30]. The DFT calculation was performed by the hybrid B3LYP method, which is based on the method proposed by Becke and a mixture of the exact (Hartree-Fock, HF) and DFT exchange utilizing the B3 functional was considered, together with the LYP correlation functional [31,32]. The B3LYP calculations were carried out using the basis set D95V++** [33]. After obtaining the converged geometry, the harmonic vibrational frequencies were calculated the same theoretical level to confirm whether the number of imaginary frequencies is zero for the stationary points. 4. Conclusions Since water solubility is an important factor in drug development, we synthesized two CPT derivatives, P210 and P211, and compared their solubilities. To rationalize the differences in their solubilities, we subjected them and their hydrated complexes to DFT-B3LYP comparison. Based on our recent computational results, the carbonyl group on ring D (Figure 1) in both P210 and P211 formed the strongest interactions with a water molecule. Although a morpholine ring is a polar group, its introduction into CPT reduced the dipole moment of CPT and did not induce large differences in the interaction strength between CPT and a water molecule. Therefore, P211 might not be more soluble in water than P210. In-vivo and in-vitro studies on the differences in their anticancer activities are in progress. Author Contributions: C.-H.L. and T.-H.C. conceived, designed experiments, analyzed the data, and wrote the paper; C.-C.C. and Y.-L.W. performed the experiments. Funding: The authors would like to thank the Ministry of Science and Technology, Taiwan (MOST 107-2113-M-039-007), and China Medical University for their financial support.
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Acknowledgments: We thank the National Center for High-performance Computing (Taiwan) for providing computing time. Special thanks are also due to the reviewers for their helpful suggestions and comments. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
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Sample Availability: Samples of the compounds 16b, P210, and P211 are available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
