Synthesis, spectroscopic, and antimicrobial activity studies of novel 10-substituted camptothecin phosphorothioate analogs

Article abstract of DOI:10.1080/10426507.2011.631643

A series of title compounds 2 and 3 were efficiently synthesized via the condensation of 10-hydroxycamptothecin with various symmetric (O,O-monoaryl)-thiophosphoryl chlorides and asymmetric (O-ethyl-O-aryl)- thiophosphoryl chlorides in sodium hydroxide powder and acetonitrile system. The structures of title compounds 2 and 3 were confirmed by elemental analysis, IR, ¹H NMR, ¹³C NMR, ³¹P[¹H] NMR,and mass spectral data. These symmetric [(O,O-monoaryl)-thiophosphoryl)]-(20S)- camptothecin (2a-f) and asymmetric [(O-ethyl-O-aryl)-thiophosphoryl)]-(20S)- camptothecin (3a-f) compounds were also tested for their in vitro antimicrobial activities against some bacterial strains, namely, S. aureus, B. Simplex, E. acetylicum, E. coli, P. aeruginosa, S. flexenari, S. aureus, S. typhi, and some fungal strains Aspergillus niger, Aspergillus flavus (molds), S. cerevisiae, C. albicans, T. longifucus, A. flavus, M. canis, F. solani, and C. glaberata (yeasts). Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the related elements to view the free supplemental file. Copyright Taylor and Francis Group, LLC 2012.

Full text of DOI:10.1080/10426507.2011.631643

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Synthesis, Spectroscopic, and
Antimicrobial Activity Studies of
Novel 10-Substituted Camptothecin
Phosphorothioate Analogs
Abdel-Nasser M. A. Alaghaz a b , Badr A. El-Sayad c & Salwa A. H.
Albohy b
a Department of Chemistry, Faculty of Science , Jazan University ,
Jazan , Saudi Arabia
b Department of Chemistry, Faculty of Science (Boys) , Al-Azhar
University , Nasr City , Cairo , Egypt
c Department of Chemistry, Faculty of Science (Girls) , Al-Azhar
University , Nasr City , Cairo , Egypt
Published online: 30 May 2012.
To cite this article: Abdel-Nasser M. A. Alaghaz , Badr A. El-Sayad & Salwa A. H. Albohy (2012)
Synthesis, Spectroscopic, and Antimicrobial Activity Studies of Novel 10-Substituted Camptothecin
Phosphorothioate Analogs, Phosphorus, Sulfur, and Silicon and the Related Elements, 187:7, 799-807,
DOI: 10.1080/10426507.2011.631643
To link to this article: http://dx.doi.org/10.1080/10426507.2011.631643
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Phosphorus, Sulfur, and Silicon, 187:799–807, 2012
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2011.631643
SYNTHESIS, SPECTROSCOPIC, AND ANTIMICROBIAL
ACTIVITY STUDIES OF NOVEL 10-SUBSTITUTED
CAMPTOTHECIN PHOSPHOROTHIOATE ANALOGS
Abdel-Nasser M. A. Alaghaz,1,2 Badr A. El-Sayad,3
and Salwa A. H. Albohy2,*
1Department of Chemistry, Faculty of Science, Jazan University, Jazan,
Saudi Arabia
2Department of Chemistry, Faculty of Science (Boys), Al-Azhar University,
Nasr City, Cairo, Egypt
3Department of Chemistry, Faculty of Science (Girls), Al-Azhar University,
Nasr City, Cairo, Egypt
GRAPHICAL ABSTRACT
Abstract A series of title compounds 2 and 3 were efficiently synthesized via the condensation
of 10–hydroxycamptothecin with various symmetric (O,Oꢁ-monoaryl)-thiophosphoryl chlorides
and asymmetric (O-ethyl-Oꢁ-aryl)-thiophosphoryl chlorides in sodium hydroxide powder and
acetonitrile system. The structures of title compounds 2 and 3 were confirmed by elemental
analysis, IR, 1H NMR, 13C NMR, 31P[1H] NMR,and mass spectral data. These symmetric
[(O,Oꢁ-monoaryl)-thiophosphoryl)]-(20S)-camptothecin (2a–f) and asymmetric [(O-ethyl-Oꢁ-
aryl)-thiophosphoryl)]-(20S)-camptothecin (3a–f) compounds were also tested for their in
vitro antimicrobial activities against some bacterial strains, namely, S. aureus, B. Simplex, E.
acetylicum, E. coli, P. aeruginosa, S. flexenari, S. aureus, S. typhi, and some fungal strains
Aspergillus niger, Aspergillus flavus (molds), S. cerevisiae, C. albicans, T. longifucus, A. flavus,
M. canis, F. solani, and C. glaberata (yeasts).
Received 17 August 2011; accepted 10 October 2011.
∗Permanent address: Department of Chemistry, Faculty of Science (Boys), Al-Azhar University, Nasr City,
Cairo, Egypt
Address correspondence to Abdel-Nasser M. A. Alaghaz, Department of Chemistry, Faculty of Science,
Jazan University, Jazan, Saudi Arabia. E-mail: aalajhaz@yahoo.com
799

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A.-N. M. A. ALAGHAZ ET AL.
Supplemental materials are available for this article. Go to the publisher’s online edition of
Phosphorus, Sulfur, and Silicon and the related elements to view the free supplemental file.
Keywords Synthesis; antimicrobial activity; camptothecine; phosphorothioate
INTRODUCTION
20(S)–Camptothecin (CPT), a pentacyclic alkaloid, originally isolated from the Chi-
nese plant Camptotheca acuminata by Wall and Wani in 1966,1 is one of the most important
lead compounds in anticancer research. The antitumor activity of CPT is thought to be due
to its ability to stabilize the reversible covalent DNA topoisomerase I (top 1) complex,2–7
preventing the relegation step of the breakage/rejoining reaction mediated by the enzyme.
The net result is that the drug causes fragmentation of chromosomal DNA, cell death and
extensive sister chromatid exchange and chromosomal aberrations.8–13 Elucidation of the
specific target and mechanisms of camptothecin has stimulated intensive efforts to iden-
tify novel analogs that overcome the drawbacks of the natural camptothecin molecule,
which include low solubility in water; severe and unpredictable toxicity, including hem-
orrhagic cystitis; reversibility of the drug–target interaction; lactone instability; and drug
resistance.14–16 One of the initial major strategies in this regard has been to improve the
solubility of the natural camptothecin by chemical modification.16,17 This approach has pro-
duced different series of water-soluble analogs or water-soluble pro-drugs, among which
topotecan and irinotecan are the most successful. CPT topoisomerase I (top I) inhibitors are
proving useful against a range of refractory tumors, most prominently against some colon
and ovarian cancers.18–20 Two of the CPTs, topotecan and CPT–11, have received Food and
Drug Administration approval, and several others are in clinical trials.
In the present paper, we would like to report the results on the antimicrobial activity
investigation of twelve representatives of symmetric 10-[(O,Oꢁ-monoaryl)thiophosphoryl]-
(20S)-camptothecin (2a–f) and asymmetric 10-[(O-ethyl-Oꢁ-aryl)thiophosphoryl-(20S)-
camptothecin (3a–f). The synthetic route is shown in Figure 1. Characterization was per-
formed by elemental analysis, IR, 1H NMR, 13C NMR, 31P NMR, and mass spectral data.
The biological activities of the synthesized comounds (2a–f) and (3a–f), have been ex-
amined against some antimicrobial strains namely, S. aureus, B. Simplex, E. acetylicum
(Gram-positive bacteria), E. coli, P. aeruginosa, S. flexenari, S. aureus, S. typhi (Gram-
negative bacteria); and A. niger, A. flavus (molds), S. cerevisiae, C. albicans, T. longifucus,
A. flavus, M. canis, F. solani, and C. glaberata (yeasts) for evaluation of antibacterial
and antifungal activities of the synthesized chemical compounds. The results obtained
were compared with standard antibiotic: Ciprofloxacin and the standard antifungal drug:
Amphotericin–B.
RESULTS AND DISCUSSION
Chemistry
The present work was to design and synthesize some new symmetric (O,Oꢁ-
monoaryl)-thiophosphoryl-(20S)-camptothecin and asymmetric [(O-ethyl-Oꢁ-aryl)-
thiophosphoryl-(20S)-camptothecin derivatives (Figure 1) carrying the biologically active
thiophosphoate moiety, and hence expected to show antimicrobial activity.

SYNTHESIS, SPECTROSCOPIC AND ANTIMICROBIAL ACTIVITY STUDIES
801
Figure 1 Scheme for synthesis of symmetric [(O,Oꢁ-monoaryl)-thiophosphoryl)]-(20S)-camptothecin (2a–f) and
asymmetric [(O—ethyl-Oꢁ-aryl)-thiophosphoryl)]-(20S)-camptothecin (3a–f) compounds.
10–hydroxycamptothecin 1 reacted with various thiophosphoryl chlorides to yield
the target compounds 2, 3 in moderate yields. In order to optimize the reaction conditions,
we scanned different bases and solvent systems, such as Et3N/CH2Cl2, Et3N/CHCl3,
Et3N/dioxane, pyridine/dioxane, pyridine/CHCl3, K2CO3/dioxane, K2CO3/CH3CN,
NaHCO3/H2O+THF, and NaOH(s)/CH3CN.
Finally, we found that the reaction gave the best results in mild reaction condi-
tions in the NaOH(s)/CH3CN system, and no byproduct was detected by TLC (thin layer
chromatography).
The structures of compounds 2a–f and 3a–f were deduced from their spectral data
(IR, 1H, 13C, and 31P NMR, MS) and elemental analyses. Selected spectra for 2a and 3a are
shown in Figures S1–6 (Supplemental Materials). IR spectra of compounds 2 and 3 showed
normal stretching absorption bands, indicating the existence of OH (∼3500 cm−1),21 C O
(∼1720 cm−1),22 C N (∼1600 cm−1),23 Ar H (∼1510, 1450 cm−1),21 P O C (∼1225,
1060, 1030 cm−1),24 and P S (∼1000 cm−1),25,26 and in 31P NMR spectra, the P signal
in all of the title compounds was displayed as a singlet, in the chemical shift range of
δ 60.4–76.6.26,27
The 1H, 13C, and 31P NMR spectra of these novel camptothecin derivatives showed
the correct characteristic proton peaks for their different subsistents. The mass spectra of
obtained compounds showed molecular ion peaks that were consistent with their molecular
formulae.

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A.-N. M. A. ALAGHAZ ET AL.
Biological Activity. In the present study, all chemically synthesized compounds
were evaluated against three Gram-positive, five Gram-negative bacteria, and nine fungi
(two mold and two strains). Minimum Inhibitory concentrations (MIC) of some of these syn-
thesized compounds against some Gram-positive bacteria were determined by the method
given by Andrews.28 The structure activity relationship (SAR) studies revealed that the pres-
ence of an electron-withdrawing group (2b, 2f, 3d, and 3f) on the O,O-monophenyl ring
(2) or O-ethylO-phenyl ring (3) increased the antimicrobial activity, and activity decreased
in the presence of electron-releasing atoms or groups.29
EXPERIMENTAL
Melting points (◦C, uncorrected) were determined in open capillaries on a Gal-
lenKemp melting point apparatus (Sanyo GallenKemp, Southborough, UK).
The infrared spectra were recorded in KBr pellets on a Pye unicam SP 3300 and
FTIR S101PC Shimadzu Infrared Spectrometers (Shimadzu, Tokyo, Japan).
NMR spectra were obtained in deuterated CDCl3 or DMSO (dimethylformamide)
on a Varian MERCURY (1H: 300 MHz and 13C: 75 MHz) spectrometer (Bruker, Flawil,
Switzerland, δ ppm) using TMS as an internal reference. 31P NMR spectra were measured
on the same spectrometer using H3PO4 (85%) as external reference.
Electron impact Mass Spectra were recorded on a Shimadzu Gc Ms Qp 5000
instrument (Shimadzu, Tokyo, Japan).
Elemental analysis was performed on Carlo Erba 1108 Elemental Analyzer (Heraeus,
Hanau, Germany). The phosphorus content was determined gravimetrically as phospho-
ammoniummolybdate.30
TLC analysis was carried out on silica gel plates GF254. Flash column chromatography
was carried out on silica gel 300–400 mesh. HPLC analysis was performed on a Waters
Alliance 2690 instrument with UV detection at 360 nm: column: Waters Symmetry–C18.
All of the solvents and materials were reagent grade and purified are required. Camp-
tothecin was purchased from Sigma Chemical (St. Louis, MO).
10–hydroxy–camptothecin (1) was prepared by following the literature procedure.31
Symmetric thiophosphoric chlorides were prepared according to the procedure in the
literature.26
Asymmetric thiophosphoric chlorides were prepared according to the procedures in
the literature.32,33
Preparation of Compounds 2, 3: General Procedure
A mixture of 10—hydroxy-CPT (0.732 g, 2 mmol), NaOH powder (0.08 g, 2 mmol)
and anhydrous acetonitrile (15 mL) were added into a three necked flask at 298 K. After
vigorously stirring for 5∼10 min, then (O,Oꢁ-monoaryl) thiophosphoryl chloride or (O-
ethyl-Oꢁ-aryl) thiophosphoryl chloride (2 mmol) in acetonitrile (5 mL) was added dropwise.
TLC was carried out to de◦tect the reaction throughout. After the mixture was stirred
under N2 atmosphere at 60 C for 72∼ 75 h, it was added to certain water. The mixture
was filtered and the solid was washed with water and acetone, and then dried. After
the solvent was removed under reduced pressure, the residue was taken up in the mixed
solution (eluent: CHCl3/CH3OH 8:2) and purified by column chromatography (eluent:
CHCl3/CH3OH 100:1–100:3).

SYNTHESIS, SPECTROSCOPIC AND ANTIMICROBIAL ACTIVITY STUDIES
803
O,Oꢀ-monophenyl-[(20S)-camptothecin]-phosphorothionate (2a). Yel-
low oil, yield: 77.3%, IR (KBr, cm−1)ν: 3500 (OH), 1721 (C O), 1600 (C N), 1510,
1
1454 (Ar), 1224, 1063, 1033 (P O C), 1000 (P S). H NMR (300 MHz, DMSO–d6)
δ: 0.99 (3H, t, J = 7.5 Hz, H–18), 1.86 (2H, m, H–19), 4.73 (2H, s, H–5), 4.40 (2H, s,
H–17), 2.54 (1H, s, 20 OH), 6.68–7.86 (8H, m, Ar H); 13C NMR (75 MHz, DMSO–d6)
δ: 7.9, 30.4, 51.6, 64.7, 72.5, 108.6, 115.2 × 2, 117.7, 118.8, 121.7 × 2, 129.9 × 2, 130.8,
136.4, 144.1, 145.4 × 2, 145.7, 148.4, 150.2, 153.4, 158.5, 172.7; 31P NMR (121.5 MHz,
DMSO–d6) δ: 76.5 (P S); MS m/z (%): 613 [M+] (2.1), 76 (100). Anal. Calcd. for
C26H18BrN2O7PS: C, 50.91; H, 2.96; Br, 13.03; N, 4.57; P, 5.05; S, 5.23. Found: C, 50.92;
H, 2.95; N, 4.55; P, 5.02; S, 5.24.
O,Oꢀ-[mono-p-bromophenyl)-(20S)-camptothecin]-phosphorothionate
(2b). Yellow oil, yield: 76.3%, IR (KBr, cm−1)ν: 3505 (OH), 1716 (C O), 1602 (C N),
1513, 1452 (Ar), 1224, 1063, 1034 (P O C), 1005 (P S). 1H NMR (300 MHz,
DMSO–d6) δ: 0.90 (3H, t, J = 7.5 Hz, H–18), 1.86 (2H,m, H–19), 2.35 (3H, t, J = 7.2 Hz,
CH3), 4.25 (2H, s, H–5), 4.76, 4.74 (2H, s, H–17), 2.55 (1H, s, 20 OH), 6.72–7.89 (7H, m,
Ar H); 13C NMR (75 MHz, DMSO–d6) δ: 7.9, 30.5, 51.7, 65.4, 72.7, 97.4, 108.3, 111.2,
115.3, 119.8, 120.2, 123.7, 126.7, 130.3 × 2, 130.9, 138.4, 144.2, 147.5, 145.7, 147.6,
149.4, 150.4, 153.2, 156.8, 172.4; 31P NMR (121.5 MHz, DMSO–d6) δ: 72.2 (P S); MS
m/z (%): 548 [M+] (1.3), 83 (100). Anal. Calcd. for C27H21N2O7PS: C, 59.12; H, 3.86; N,
5.11; P, 5.65; S, 5.85. Found: C, 59.10; H, 3.84; N, 5.10; P, 5.63; S, 5.82.
O,Oꢀ-[(mono-4-tolyl)]-[(20S)-camptothecin]-phosphorothi-onate
(2c).
Yellow oil, yield: 65.8%, IR (KBr, cm−1)ν: 3508 (OH), 1717 (C O), 1602 (C N), 1513,
1452 (Ar), 1225, 1064, 1036 (P O C), 1002 (P S). 1H NMR (300 MHz, DMSO–d6) δ:
0.74 (3H, t, J = 7.5 Hz, H–18), 1.44 (2H,m, H–19), 2.27 (3H, t, J = 7.2 Hz, CH3), 5.25
(2H, s, H–5), 5.43 (2H, s, H–17), 6.54 (1H, s, 20-OH), 7.17–7.89 (7H, m, Ar H); 13C
NMR (75 MHz, DMSO–d6) δ: 7.9, 11.5, 19.6, 30.7, 51.3, 56.9, 65.7, 73.0, 97.6, 108.3,
114.2, 119.8, 120.3, 122.7, 125.9, 130.2, 130.9, 132.7, 138.3 142.2, 145.3, 145.5, 149.8,
150.6, 153.3; 31P NMR (121.5 MHz, DMSO–d6) δ: 72.3 (P S); MS m/z (%): 548 [M+]
(1.3), 83 (100). Anal. Calcd. for C27H21N2O7PS: C, 59.12; H, 3.86; N, 5.11; P, 5.65; S,
5.85. Found: C, 59.10; H, 3.84; N, 5.10; P, 5.63; S, 5.82.
O,Oꢀ-[(mono-3,4-dimethylphenyl)]-[(20S)-camptothecin] Phosphorothi-
onate (2d). Yellow oil, yield: 55.3%, IR (KBr, cm−1)ν: 3499 (OH), 1719 (C O), 1602
(C N), 1514, 1450 (Ar), 1220, 1060, 1035 (P O C), 1004 (P S). 1H NMR (300 MHz,
DMSO–d6) δ: 0.88 (3H, t, J = 7.4 Hz, H–18), 1.86 (2H, m, H–19), 2.37 (3H, s, CH3), 2.39
(3H, m, CH3), 5.23 (2H, s, H–5), 5.42 (2H, s, H–17), 6.53 (1H, s, 20 OH), 7.18–7.89 (6H,
m, Ar H); 13C NMR (75 MHz, DMSO–d6) δ: 7.9, 11.5, 19.6, 30.7, 51.3, 56.9, 65.7, 73.0,
97.6, 108.3, 114.2, 119.8, 120.3, 122.7, 125.9, 130.2, 130.9, 132.7, 138.3 142.2, 145.3,
145.5, 149.8, 150.6, 153.3; 31P NMR (121.5 MHz, DMSO–d6) δ: 72.1 (P S); MS m/z (%):
562 [M+] (3.2), 104 (100). Anal. Calcd. for C28H23N2O7PS: C, 59.78; H, 4.12; N, 4.98; P,
5.51; S, 5.70. Found: C, 59.79; H, 4.11; N, 4.97; P, 5.50; S, 5.71.
O,Oꢀ-[(mono-4-tertiarybutylphenyl)]-[(20S)-camptothecin]-phosphoro-
thionate (2e). Yellow oil, yield: 76.5%, IR (KBr, cm−1)ν: 3500 (OH), 1718 (C O),
1606 (C N), 1514, 1453 (Ar), 1225, 1062, 1033 (P O C), 1001 (P S). 1H NMR (300
MHz, DMSO–d6) δ: 0.88 (3H, t, J = 7.2 Hz, H–18), 1.85 (2H, m, H–19), 2.03 [6H, d,
CH (CH3)2], 2.21 (H, m, CH3CHCH3), 5.13 (2H, s, H–5), 5.24 (2H, s, H–17), 2.46 (1H,
s, 20 OH), 6.98–7.78 (8H, m, Ar H); 13C NMR (75 MHz, DMSO–d6) δ: 7.2, 30.3, 31.
5 × 3, 34.4, 51.3, 65.7, 72.8, 97.7, 108.4, 109.0, 119.6, 120.3, 121.3, 122.4, 130.3 × 2,
130.8, 138.2, 139.3, 141.2, 144.0, 144.6, 145.5, 149.4, 150.3, 153.8, 156.7, 172.6; 31P

804
A.-N. M. A. ALAGHAZ ET AL.
NMR (121.5 MHz, DMSO–d6) δ: 71.7 (P S); MS m/z (%): 576 [M+] (0.8), 77 (100).
Anal. Calcd. for C29H25N2O7PS: C, 60.41; H, 4.37; N, 4.86; P, 5.37; S, 5.56. Found: C,
60.40; H, 4.38; N, 4.87; P, 5.36; S, 5.57.
O,Oꢀ-[(mono-4-chlorophenyl)]-[(20S)-camptothecin]-phos-phorothio-nate
(2f). Yellow oil, yield: 79.3%, IR (KBr, cm−1) ν: 3500 (OH), 1725 (C O), 1610
(C N), 1515, 1455 (Ar), 1225, 1060, 1035 (P O C), 1005 (P S). 1H NMR (300 MHz,
DMSO–d6) δ: 0.92 (3H, t, J = 7.3 Hz, H–18), 1.89 (2H, m, H–19), 4.58 (2H, s, H–5),
4.76,4.73 (2H, s, H–17), 2.63 (1H, s, 20 OH), 6.66–7.82 (8H, m, Ar H); 13C NMR (75
MHz, DMSO–d6) δ: 8.2, 30.9, 51.4, 65.7, 72.6, 97.5, 108.8, 111.6, 115.5, 119.4, 120.8,
123.9, 126.6, 130.4 × 2, 131.2, 138.9, 144.8, 147.7, 145.9, 147.8, 149.7, 150.7, 153.6,
156.9, 172.8; 31P NMR (121.5 MHz, DMSO–d6) δ: 76.6 (P S); MS m/z (%): 569 [M+]
(1.2), 76 (100). Anal. Calcd. for C26H18ClN2O7PS: C, 54.89; H, 3.19; Cl, 6.23; N, 4.92; P,
5.44; S, 5.64. Found: C, 54.87; H, 3.18; Cl, 6.24; N, 4.93; P, 5.43; S, 5.65.
O-ethyl-O-phenyl-[(20S)-camptothecin]-phosphorothionate (3a). Yellow
powder, yield: 65.0%, IR (KBr, cm−1) ν: 3488 (OH), 1723 (C O), 1612 (C N), 1512,
1455 (Ar), 1227, 1065, 1034 (P O C), 1007 (P S). 1H NMR (300 MHz, DMSO–d6) δ:
0.86 (3H, t, J = 7.3 Hz, H–18), 1.86 (2H, m, H–19), 4.22 (2H, s, H–5), 4.42, 4.44 (2H,
s, H–17), 2.05 (1H, s, 20–OH), 4.09 (2H, s, H–22), 1.29 (3H, s, H–23), 6.68–7.89 (8H,
m, Ar H); 13C NMR (75 MHz, DMSO–d6) δ: 6.8, 17.7, 30.2, 51.2, 63.5, 65.7,72.5, 78.5,
109.3, 120.2, 120.7 × 2, 121.4, 130.2 × 2, 130.6 × 2, 144.1, 145.5, 149.3, 150.5, 151.4,
153.6, 156.8, 174.0; 31P NMR (121.5 MHz, DMSO–d6) δ: 62.7 (P S); MS m/z (%): 550
[M+] (0.7), 83 (100). Anal. Calcd. for C28H27N2O6PS: C, 61.08; H, 4.94; N, 5.09; P, 5.63;
S, 5.82. Found: C, 61.04; H, 4.92; N, 5.07; P, 5.61; S, 5.80.
O-ethyl-O-(4-Tolyl)-[(20S)–camptothecin)]-phosphorothi-onate
(3b).
Yellow powder, yield: 86.3%, IR (KBr, cm−1)ν: 3500 (OH), 1719 (C O), 1608 (C N),
1519, 1454 (Ar), 1227, 1060, 1035 (P O C), 1009 (P S). 1H NMR (300 MHz,
DMSO–d6) δ: 0.93 (3H, t, J = 7.0 Hz, H–18), 1.82 (2H, m, H–19), 2.33 (3H, s, Ar CH3),
4.28–4.34 (2H, m, CH2CH3), 4.21 (2H, s, H–5), 4.42 (2H, s, H–17), 2.49 (1H, s, 20–OH),
6.92–7.94 (8H, m, Ar H); 13C NMR (75 MHz, DMSO–d6) δ: 6.6, 17.4, 22.6, 30.5, 51.6,
63.7, 65.4, 72.3, 78.4, 109.4, 120.3, 118.7 × 2, 121.7, 130.4 × 2, 130.4 × 2, 144.3,
145.7, 149.7, 150.7, 151.7, 153.9, 156.7, 174.2; 31P NMR (121.5 MHz, DMSO–d6) δ: 60.4
(P S); MS m/z (%): 564 [M+] (1.7), 91 (100). Anal. Calcd. for C29H29N2O6PS: C, 61.69;
H, 5.18; N, 4.96; P, 5.49; S, 5.68. Found: C, 61.64; H, 5.16; N, 4.93; P, 5.45; S, 5.64.
O-ethyl-O-[(4-methoxyphenyl)]-[(20S)-camptothecin]-phosphorothio-
nate (3c). Yellow powder, yield: 78.9%, IR (KBr, cm−1)ν: 3485 (OH), 1719 (C O),
1602 (C N), 1511, 1452 (Ar), 1223, 1062, 1030 (P O C), 1002 (P S). 1H NMR (300
MHz, DMSO–d6) δ: 0.89 (3H, t, J = 7.0 Hz, H–18),
1.86 (2H, m, H–19), 3.83 (3H, s, ArOCH3), 4.27–4.36 (2H, m, CH2CH3), 4.43 (2H,
s, H–5), 4.48 (2H, s, H–17), 2.50 (1H, s, 20–OH), 6.78–7.87 (9H, m, Ar–H); 13C NMR
(75 MHz, DMSO–d6) δ: 6.83, 17.4, 30.5, 51.6, 55.6, 63.7, 65.4, 72.3, 78.4, 109.4, 115.5 ×
2, 117.5, 120.3, 122.5, 124.6, 130.3 × 2, 144.3, 145.7, 149.7, 150.3, 151.2, 153.4, 156.7,
173.4; 31P NMR (121.5 MHz, DMSO–d6) δ: 60.7 (P S); MS m/z (%): 580 [M+] (2.3), 45
(100). Anal. Calcd. for C29H29N2O7PS: C, 59.99; H, 5.03; N, 4.83; P, 5.33; S, 5.52. Found:
C, 59.97; H, 5.06; N, 4.87; P, 5.35; S, 5.54.
O-ethyl-O-[(4-chlorophenyl)]-[(20S)-camptothecin]-phosphorothio-nate
(3d). Yellow powder, yield: 77.5%, IR (KBr, cm−1)ν: 3502 (OH), 1720 (C O), 1600
(C N), 1510, 1458 (Ar), 1224, 1063, 1032 (P O C), 1000 (P S). 1H NMR (300 MHz,
DMSO–d6) δ: 0.87 (3H, t, J = 6.5 Hz, H–18), 1.82 (2H, m, H–19), 4.78–4.97 (2H, m,

SYNTHESIS, SPECTROSCOPIC AND ANTIMICROBIAL ACTIVITY STUDIES
805
CH2CH3), 4.25 (2H, s, H–5), 4.47 (2H, s, H–17), 2.52 (1H, s, 20 OH), 6.66–7.73 (8H,
m, Ar H); 13C NMR (75 MHz, DMSO–d6) δ: 7.7, 17.7, 22.8, 30.7, 51.5, 63.8, 65.9, 72.8,
78.6, 109.7, 120.6, 118.6 × 2, 121.9, 125.7 × 2, 132.4 × 2, 130.7 × 2, 145.2, 145.9, 149.5,
150.8, 152.2, 154.0, 157.8, 174.7; 31P NMR (121.5 MHz, DMSO–d6) δ: 67.3 (P S); MS
m/z (%): 585 [M+] (3.1), 111 (100). Anal. Calcd. for C28H26ClN2O6PS: C, 57.49; H, 4.48;
Cl, 6.06; N, 4.79; P, 5.29; S, 5.48. Found: C, 57.44; H, 4.42; Cl, 6.03; N, 4.76; P, 5.26; S,
5.43.
O-ethyl-O-[(4-nitrophenyl)]-[(20S)-camptothecin]-phosphorothionate
(3e). Yellow powder, yield: 82.6%, IR (KBr, cm−1)ν: 3502 (OH), 1720 (C O), 1600
(C N), 1510, 1458 (Ar), 1224, 1063, 1032 (P O C), 1010 (P S). 1H NMR (300 MHz,
DMSO–d6) δ: 0.96 (3H, t, J = 7.2 Hz, H–18), 1.89 (2H, m, H–19), 4.74–4.95 (2H, m,
CH2CH3), 4.46 (2H, s, H–5), 4.47 (2H, s, H–17), 2.65 (1H, s, 20 OH), 6.69–7.96 (8H,
m, Ar H); 13C NMR (75 MHz, DMSO–d6) δ: 7.6, 17.4, 22.6, 30.3, 51.4, 63.5, 65.3,
72.4, 78.3, 109.2, 120.4, 121.3 × 2, 125.7 × 2, 130.8, 132.4 × 2, 140.3, 141.5, 145.7,
149.8, 150.7, 152.2, 154.0, 156.7, 157.2, 174.7; 31P NMR (121.5 MHz, DMSO–d6) δ:
65.7 (P S); MS m/z (%): 595 [M+] (5.6), 122 (100). Anal. Calcd. for C28H26N3O8PS: C,
56.47; H, 4.40; N, 7.06; P, 5.20; S, 5.38. Found: C, 56.44; H, 4.42; N, 7.04; P, 5.21; S, 5.33.
O-ethyl-O-[(2,4-dichlorophenyl)]-[(20S)-camptothecin]-phosphoroth-io
nate (3f). Yellow powder, yield: 79.8%, IR (KBr, cm−1)ν: 3500 (OH), 1721 (C O),
1601 (C N), 1512, 1455 (Ar), 1225, 1062, 1037 (P O C), 1009 (P S). 1H NMR (300
MHz, DMSO–d6) δ: 1.03 (3H, t, J = 7.0 Hz, H–18), 1.86 (2H, m, H–19), 4.78 (2H, m,
CH2CH3), 4.22 (2H, s, H–5), 4.75, 4.78 (2H, s, H–17), 2.62 (1H, s, 20–OH), 6.78–7.97
(8H, m, Ar H); 13C NMR (75 MHz, DMSO–d6) 8.4, 18.6, 22.4, 30.6, 52.5, 63.7, 65.7,
73.2, 78.9, 109.8, 120.5, 122.3, 14.8, 129.4, 132.3, 132.3 × 2, 136.7, 142.1, 143.5, 145.6,
150.3, 150.9, 151.7, 153.7, 155.4, 156.3, 173.2; 31P NMR (121.5 MHz, DMSO–d6) δ: 66.8
(P S); MS m/z (%): 619 [M+] (3.2), 146 (100). Anal. Calcd. for C28H25Cl2N2O6PS: C,
54.29; H, 4.07; Cl, 11.45; N, 4.52; P, 5.00; S, 5.18. Found: C, 54.28; H, 4.03; Cl, 11.43; N,
4.50; P, 5.02; S, 5.15.
Pharmacology
Test Microorganisms. Sixteen microbial strains (four bacterial and four fungi)
were selected on the basis of their clinical importance in causing diseases in humans.
S. aureus, B. Simplex, E. acetylicum (Gram-positive bacteria), E. coli, P. aeruginosa, S.
Flexenari, S. typhi (Gram-negative bacteria), andA. niger, A. flavus (molds), S. cerevisiae, C.
albicans, T. longifucus, A. flavus, M. canis, F. solani, C. glaberata (yeasts) were screened for
evaluation of antibacterial and antifungal activities of the synthesized chemical compounds.
In vitro Antimicrobial Activity
Antibacterial and Antifungal Assays. The agar diffusion method34 was fol-
lowed for antibacterial and antifungal susceptibility tests.
Determination of Minimum Inhibitory Concentration (MIC). The MIC of
the chemically synthesized compound was tested against bacterial and yeast strains through
a macro dilution tube method.27
In vitro Antimicrobial Activity (for Molds)35. All the newly synthesized com-
pounds were evaluated for their antimicrobial activity (molds) by poison food technique.36
All the test molds were grown on Sabouraud dextrose agar (SDA) at 25 ◦C for 7–8 days.

806
A.-N. M. A. ALAGHAZ ET AL.
One-week-old culture of the mold was used as inoculum for evaluating antifungal activ-
◦
ity of chemical compounds. The molten SDA (45 C) was poisoned by the addition of
100 mL volume having concentration of 1.0 mg/mL of each compound reconstituted in
the DMSO and poured into the sterile Petri plates. The prepared SDA plates containing
the test compound were inoculated with fungal plugs (8 mm diameter) obtained from the
actively growing margins of the fungal plates. Plates were incubated at 25 ◦C for 7 days.37
The medium with DMSO as solvent was used as a negative control whereas media with
Amphotericin-B (standard antifungal drug) were used as positive control.
The experiments were performed in triplicates. Diameter of fungal colony was mea-
sured and expressed as percentage inhibition and determined by the formula given below:
Percentage inhibition of mycelial growth = (dc – dt)/dc × 100
dc = average fungal colony diameter in control sets
dt = average fungal colony diameter in treatment sets
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