Design, synthesis, docking studies and biological evaluation of novel dihydro-1,3,5-triazines as human DHFR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.11.010

A novel series of dihydro-1,3,5-triazine derivatives bearing a heteroatom spiro-ring were designed and synthesized on the basis of molecular flexible docking work, and their biological activities were evaluated. Compounds A2, A5, B1 and B3 showed potent human dihydrofolate reductase (hDHFR) inhibitory activity with IC₅₀values of 7.46 nM, 3.72 nM, 6.46 nM, 4.08 nM, versus reference drug methotrexate (MTX). From the molecular docking result we concluded that the conformation space generated by deformation of the flexible residue Phe31 is favorable for the binding of the spiro-ring, and inserting heteroatom into spiro ring might increase the binding affinity. There were 24 compounds with broadspectrum antiproliferative activity against several tumor cell lines (HCT116, A549, HL-60, HepG2 and MDA-MB-231) with IC₅₀values ranging from 0.79 to 0.001 μM. The antitumor activity in?vivo of compound A2 was determined in a human alveolar basal epithelial cell line A549 xenograft model. This study offered novel anticancer agents with high inhibitory activity that target hDHFR and have a binding mode of the novel molecular scaffold with hDHFR. This provides potent support for further development of novel hDHFR inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.11.010

Accepted Manuscript
Design, synthesis, docking studies and biological evaluation of novel dihydro-1,3,5-
triazines as human DHFR inhibitors
Xiao-Tian Zhou, Kuai-Le Lin, Xiang Ma, Wai-Keung Chui, Wei-Cheng Zhou
PII:
S0223-5234(16)30948-5
10.1016/j.ejmech.2016.11.010
EJMECH 9043
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 October 2016
Revised Date: 4 November 2016
Accepted Date: 5 November 2016
Please cite this article as: X.-T. Zhou, K.-L. Lin, X. Ma, W.-K. Chui, W.-C. Zhou, Design, synthesis,
docking studies and biological evaluation of novel dihydro-1,3,5-triazines as human DHFR inhibitors,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.010.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Our reference: EJMECH 9043
Article reference: EJMECH_EJMECH-D-16-01957
Article title: Design, synthesis, docking studies and biological evaluation of novel
dihydro-1,3,5-triazines as human DHFR inhibitors
To be published in: European Journal of Medicinal Chemistry
Author(s): Xiao-Tian Zhoua, Kuai-Le Lina, Xiang Mab, Wai-Keung Chuic, Wei-Cheng Zhoua
aShanghai Key Lab. of Anti-infectives, State Key Lab. of New Drug and Pharmaceutical Process.
Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical
Industry,Shanghai 201203, China
bSchool of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology (HUS
T), Wuhan, 430030, Hubei, China
cDepartment of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4,
Singapore, Singapore 117543
*Corresponding author: Wei-Cheng Zhou
Email: zhouweicheng58@163.com & profzhouweicheng58@163.com
Tel: 0086-021-20572000-5025

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
1. Introduction
Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism. It converts dihydrofolate (DHF) to
tetrahydrofolate (THF), which is essential for purine and thymidylate (TMP) synthesis in cell proliferation[1]. Depressing
DHFR activity results in THF deficiency and disruption of subsequent DNA replication and resulting in cell death[2]. Therefore,
human DHFR (hDHFR) has long been a vital target enzyme in the development of antitumor chemotherapeutic agents[3].
Several hDHFR inhibitors have been successfully used in clinical oncology such as methotrexate (MTX) and trimetrexate
(TMQ). Analysis of the structure of these hDHFR inhibitors found that most of them contain a crucial nucleus: a planar bicyclic
ring consisted of nitrogen-heterocyclics substituted with amino groups such as the diaminopteridine ring of MTX, the
quinazoline ring of trimetrexate (TMQ), pyrrolo[2,3-d]pyrimidine ring of pemetrexed [4-11]. Therefore, they have similar
binding mode with hDHFR, in which, e.g., N8 of the pteridine ring of MTX contacts with Glu30 and Trp24 through a water
molecule, the 4-amino group forms hydrogen bonding with Ile7, Val115, Tyr121 and NADPH[12] (Figure 3, A).
In our previous studies, a series of 1,3,5-triazine compounds with spiro bicyclic ring were designed and synthesized, and
their biological activities were evaluated[13-14]. The results showed that some compounds, e.g., compound M0 (fig. 1) showed
potent anti-folate activity against mammalian DHFR and in vitro anti-tumor activities against human alveolar basal epithelial
cell line (A549)[14], which is comparable to MTX. The results showed that 1,3,5-triazine with spiro bicyclic ring was a novel
molecular scaffold for hDHFR inhibitors. This prompted us to undertake further investigation on new spiro-triazine derivatives
with higher anticancer activity.
In this work, we analyzed the interactions of the planar bicyclic ring of hDHFR inhibitors with the residues in the hDHFR
active site observed in several X-ray complexes (PDB ID: 1U72, 1DLS, 1KMS, 1OHK, 1S3U, 2W3B, 3NTZ) [15-22]. All
showed an anti-folate binding mode in which heteroatom and amino groups contact with residues Ile7, Trp24, Glu30, Val115,
Tyr121 and NADPH. Then, the molecular docking work of compound M0 was performed with the Flexible Docking
program[24-25]. The molecular docking data suggested that we insert oxygen and sulfur atom separately into the spiro ring of
compound M0 to create more favorable interactions. A novel series of 1,3,5-triazine derivatives bearing 9-oxaspiro or 9-
thiaspiro were designed and synthesized, and their biological activities were evaluated.
2. Results and discussion
2.1 Molecular docking studies
The molecular docking study was performed by using Flexible Docking protocol[26] in Discovery Studio 3.0. All the X-
ray complexes were extracted from the Brookhaven Protein Database (PDB http://www.rcsb.org/pdb). The structure of hDHFR
derived from the complex 1U72[4] was prepared in Discovery Studio 3.0 by removal of the original ligand MTX and
preservation of co-factor NADPH, and the hydrogen atoms and CHARMm force fields were then added. The entire hDHFR
enzyme was defined as a receptor and the site sphere was selected based on the ligand binding location within a radius of 10.0 Å.
Other parameters were set as default. For compound M0 and its derivatives, energy minimization used the ‘Minimize Ligands’
protocol.
After a superimposition work of the hDHFR proteins from X-ray complexes involving 1U72, 1DLS, 1KMS, 1OHK, 1S3U,
2W3B and 3NTZ (Figure 2), a significant conformational change in Glu31 was found seen in a ternary complex of the
furopyrimidine derivative and hDHFR[23] (PDB ID: 3NXV) (Fig. 2). This allowed us to hypothesize that the volume of key
active-site pocket could be further enlarged due to the shift of flexible residue Glu31. Thus, the residue Glu31 was identified and
selected as the flexible amino acid residue for this docking. At first, a flexible docking of original ligand MTX was carried out
to demonstrate the reliability of this model for hDHFR (RMSD to the X-ray < 1Å). Subsequently, compound M0 and its
derivatives were added. After completing the molecular docking procedure, docking poses were scored and selected based on
calculated -CDOCKER energy. We exhibited the docking pose of M0 with the best -CDOCKER energy in Figure 3B. Figures
2&3 were prepared by Discovery Studio 3.0.
In the docking results (Figure 3B), compound M0 generated the key hydrogen bonds with hDHFR in the catalytic domain.
The 4-amino group of diaminotriazine ring formed hydrogen bonds with residues Ile7, Val115 and Tyr121; the 2-amino group
of diaminotriazine ring made contacts with the residue Glu30, and the triazine ring appeared at the same position as
diaminopteridine ring of MTX. The phenoxypropyl side chain reached the hydrophobic pocket. These interactions followed the
rule of normal binding mode reported in X-ray complex 1U72. In contrast with the MTX-binding mode, the spiro-ring of M0
with the unique steric hindrance was placed at the new extended space caused by the shift in the flexible PHE31, closer to Glu30
and Trp24 than N8 of the pteridine ring of MTX. However, no possible hydrogen bond existed since the absence of electron-rich
atoms on the spiro ring for M0. The docking result of the target compound A2 bearing 9-oxaspiro indicated a homologous
binding pattern as that of M0 (Figure 4). The oxygen atom 3.60 Å from Glu 30 and 4.89 Å from Trp24, while the distances in
MTX from 1U72 complex were 4.01 Å and 6.06 Å respectively. These docking results explain the similar binding mode in the
bicyclic region from that of MTX and suggested that inserting oxygen or sulfur atom on spiro-ring might be a promising
approache to enhance the binding affinity with hDHFR by generating stronger hydrogen bonds with Glu30 and Trp24 via water-
mediated manner (or directly).

ACCEPTED MANUSCRIPT
2.2 Chemistry
Target compounds A1-A16 and B1-B12 were synthesized following the steps in Schemes 1. The 3-substituted
phenoxypropyl bromides were obtained via alkylation of commercially available substituted-phenols with K2CO3 as a base and
acetonitrile as a solvent. The synthesis of the required key intermediates 2,4-diamino-5-hydroxy-1,3,5-triaza-9-oxa-
spiro[5.5]undeca-1,3-diene hydrochloride (A0) followed the procedure reported in our previous publication[8,9]. Compound A0
was treated with NaOH and 3-substituted phenoxypropyl bromide in DMF to obtain the corresponding 2,4-diamino-5-(3'-
(substituted phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A1-A16). 3-substituted
phenoxypropyl bromides were treated with benzohydroxamic acid and sodium hydroxide in ethanol under reflux to get the
corresponding N-benzoyl-protected substituted phenoxypropyloxy hydroxylamine hydrochlorides. Next, the hydrolysis was
performed to synthesize substituted phenoxypropyloxy hydroxylamine hydrochlorides (C1-C12) by hydrochloric acid. Reaction
of the substituted phenoxypropyloxy hydroxylamine hydrochlorides (C1-C12) and cyanoguanidine for 5 hours provided the
biguanide hydrochlorides. They in reaction solution without isolation was directly subjected to HCl-catalyzed
cyclocondensation with tetrahydro-4-thiopyrone in a one pot reaction. After 5 to 15 days, final desired 2,4-diamino-5-(3'-
(substituted phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-dienes hydrochloride (B1-B12) were obtained.
2.3 hDHFR inhibitory activity
All target compounds were evaluated for the inhibitory activities against human DHFR by the reported procedure [14, 27,
28]. The results are shown in Table 1. In the inhibition assay, twenty compounds showed favorable inhibition at 5 µM, which is
superior to both leading compound M0 and MTX. Thirteen compounds (A1-A10, B1-B3) with an inhibition ratio over 90% at 5
µM were chosen for the further evaluation of IC50 values, and the others (A11-A16, B4-B12) with inhibition ratios below 90%
at 5 µM were arranged to test hDHFR inhibition ratio at a concentration of 50 µM. At 50 µM, nine compounds showed 88.2% to
94.4% inhibition ratio superior to the leading compound M0 (87.6% inhibition ratio); four compounds showed 89.8% to 93.8%
inhibition superior to MTX (89.7% inhibition ratio).
The activities of thirteen target compounds (A1-A10, B1-B3) were reported as IC50 values. Eleven compounds among them
showed the IC50 values of 46.62 to 3.72 nM, which is superior to M0 (IC50=49.04 nM), and four (A2, A5, B1,and B3) showed
favorable hDHFR inhibitory activities with IC50 value of 7.46 nM, 3.72 nM, 6.46 nM, 4.08 nM—superior to MTX (IC50=6.67
nM). In comparison to M0 (IC50=49.04 nM), 4-chloro substituted compound A6 (IC50=33.56 nM) and B2 (IC50=18.28 nM)
bearing 9-oxaspiro ring or 9-thiaspiro ring exhibited increased activities showing that insertion of oxygen and sulfur atom into
the spiro-ring could increase the activity. Furthermore, when comparing B2 (IC50=18.28 nM), B1 (IC50=6.46 nM), and B3
(IC50=4.08 nM) with A6 (IC50=33.56 nM), A9 (IC50=32.49 nM), and A2 (IC50=7.46 nM), respectively, it implied that
compounds of the thiaspiro series were generally more active than compounds of the oxaspiro series.
2.4 In vitro antiproliferative activity
The anti-tumor potency was carried out by the MTT assay [29,30] for all the target compounds against three human tumor
cell lines including human colorectal cancer cell line (HCT116), human alveolar basal epithelial cell line (A549), and human
leukemia cell line (HL-60). Twenty-one compounds showed better activity against HCT116 cells with IC50 values ranging from
0.69 ꢀM to 0.001 ꢀM relative to the positive control MTX. Thirteen compounds showed better activity against A549 cells with
IC50 values ranging from 0.24 ꢀM to 0.001 ꢀM relative to MTX. Twenty-two compounds showed activity against HL-60 cells
with IC50 values ranged from 0.83 ꢀM to 0.03 ꢀM, which is superior to MTX. In addition, compounds A10-A16, B1-B12 were
also evaluated anti-tumor activity against liver hepatocellular cell line (HepG2) and metastatic breast cancer cell line (MDA-
MB-231). Eleven compounds showed activity against HepG2 cells with IC50 values ranged from 0.38 ꢀM to 0.05 ꢀM superior to
that of MTX, and 20 compounds showed activity against MDA-MB-231 cells with IC50 values ranging from 3.57 ꢀM to 0.001
ꢀM, which is superior to MTX. Consistent with the hDHFR inhibitory activity data, there were improvements in the anti-tumor
activity due to introducing oxygen and sulfur atom. The activity of the thiaspiro series surpassed that of the oxaspiro series.
2.5 In vivo anti-tumor activity
The in vivo anti-tumor efficacy of compound A2 was evaluated in male BALB/c nude mice bearing A549 cells, according
to the published protocol[31,32]. The MTX (2 mg/kg) and 50 mg/kg compound A2 were injected i.p. 14 times over a 22 days.
Body weights were recorded per day. The tumor sizes were measured using calipers, and tumor volumes were calculated by the
formula A×B2/2 where A and B are the larger and smaller diameter of the tumor, respectively. Tumor volume (TV) was
calculated by the following formula: TV 1/2×a×b2. Relative tumor volume (RTV) was calculated by the following formula:
RTV = Vt/Vo (Vo = TV at day 1). An evaluation index of the anti-tumor effect was tumor growth inhibition rate calculated by
the following formula: Tumor growth inhibition rate (%) = (1-RTVtest / RTVcontrol)×100%. Table 2 shows the relative tumor
volume and tumor growth inhibition rate of different treatment groups. Compound A2 was identified to possess good in vivo
anti-tumor effect with 36.80% tumor growth inhibition at day 22 (for MTX, 50.84%). There was no correlation between in vitro
and in vivo activities, which was probably due to the pharmacokinetic properties.

ACCEPTED MANUSCRIPT
Table 3 describes the change in body weight of the nude mice. There was a smaller decrease in mice weight in the A2-
treated group than the MTX-treated group.
3. Conclusion
In this research, we describe a molecular docking study for compound M0 from our previous report. The results implied
that the possible extended space that results from the shift of the flexible residue Phe31 might be favorable for the binding of
the spiro-ring to the active site of hDHFR. A novel series of dihydro-1,3,5-triazine derivatives bearing a heteroatom spiro-ring
were designed and synthesized on the basis of a hypothesis from molecular flexible docking. All compounds exhibited hDHFR
inhibitory activity and anti-proliferative activity against tumor cell lines (HCT116, A549, HL-60, HepG2, and MDA-MB-231).
Compounds A2, A5, B1, and B3 showed potent hDHFR inhibitory activity with IC50 values of 7.46 nM, 3.72 nM, 6.46 nM,
and 4.08 nM, compared with reference drug MTX. 24 Compounds showed in vitro antiproliferative activity toward several
tumor cell lines with IC50 values ranging from 0.79 to 0.001 ꢀM—better than MTX. The further in vivo anti-tumor study
showed that compound A2 could inhibit tumor growth in a nude mouse A549 model. The results showed that insertion of
oxygen and sulfur atom to the spiro-ring could maintain or increase the hDHFR inhibition.
4. Experimental section
4.1 General
All target compounds were characterized on the basis of 1H NMR and 13C NMR spectroscopic data (Bruker Avance III-400
MHz and 600 MHz, respectively). Chemical shifts are reported in ppm using tetramethylsilane (TMS) as an internal standard.
the peak patterns were described as: (br) broad, (s) singlet, (d) doublet, (t) triplet, (q) quartet, and (m) multiplet. Mass spectra
were recorded with a Q-TOF mass spectrometer using electrospray ionization (ESI). A.R. grade solvents were directly used and
further purification or degas of the solvents was not required. HPLC analysis was done on Agilent HPLC system (model: 1260)
equipped with a DAD detector using Agilent Eclipse Plus C18 (5um, 4.6×150 mm) column using ACN: 0.1% phosphoric acid
aqueous solution mobile phase by gradient elution at flow rate of 1 ml/min.
4.2 General Procedure for the Synthesis of A1-A16 series
2,4-Diamino-5-hydroxy-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrochloride (0.5g, 0.002 mol) was dissolved in 20
ml of methanol, 1 molar equivalent of NaOH was added into the solution , and the mixture was refluxed for 30 min. After being
cooled to room temperature, the solvent was evaporated using vacuum, the dry white precipitate was obtained. The precipitate
was dissolved in 5 ml of DMF. 1.2 molar equivalent of 3-aryloxypropyl bromide was added. The mixture was stirred at room
temperature and TLC was used to monitor the reaction. The solution was adjusted to pH 1 using concentrated HBr, when
reaction was completed. Evaporate the solution at room temperature to remove DMF. The residue was filtered , followed by
recrystallization in 90% EtOH. Compounds A1-A16 were prepared in this method.
4.2.1 2,4-Diamino-5-(3'-phenoxypropyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A1)
1
White solid yield: 36.5%, mp: 206-208 °C; H NMR (400 MHz, DMSO-d6): δ 1.69-1.66(d,2H, J=12.8), 2.03(br, s, 2H),
2.18-2.14(dd, 2H, J=6), 3.59-3.54(t, 2H, J=11.6), 3.78-3.74(dd, 2H, J1=4.8, J2=12), 4.11-4.06(dd, 4H, J1=6, J2=12), 6.97-6.93(m,
3H), 7.31-7.28(dd, 2H, J1=7.2, J2=8.8), 7.81(s, br, 1H, ex), 8.09(s, 1H, ex), 8.66(s, 1H, ex), 9.50(s, 1H, ex); 13C NMR (151 MHz,
DMSO) δ 161.60, 157.17, 134.59, 130.20, 129.39, 128.86, 79.61, 72.27, 62.71, 33.38, 32.08; HRMS calcd. for C16H23N5O3 [M-
HBr+H]+: 334.1874. found: 334.1871.
4.2.2 2,4-Diamino-5-(3'-(2'',4''-dichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A2)
1
White solid yield: 32.1%, mp: 220-222 °C; HPLC: 98.69% (tR=18.189 min); H NMR (400 MHz, DMSO-d6): δ 1.68-1.65
(d, 2H, J=12.8), 2.00(br, s, 2H), 2.22-2.19(t, 2H, J=6), 3.60-3.55(t, 2H, J=12), 3.75-3.71(dd, 2H, J1=4.4, J2=12), 4.10-4.07(t, 2H,
J=6.4), 4.20-4.17(t, 2H, J=6), 7.21-7.19(d, 1H, J=8.8), 7.38-7.35(dd, 1H J1=2.4, J2=8.8), 7.55-7.54(d, 1H, J=2.4), 7.83(s, br, 1H,
ex), 8.11(s, 1H, ex), 8.66(s, 1H, ex), 9.37(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.24, 156.60, 152.85, 129.35, 128.29,
124.76, 122.56, 115.35, 74.34, 71.76, 65.505, 62.38, 31.47, 33.60, 30.47, 26.97; HRMS calcd. for C16H21Cl2N5O3 [M-HBr+H]+:
402.1094. found: 402.1100.
4.2.3 2,4-Diamino-5-(3'-(4''-fluoro-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A3)
1
White solid yield: 25.8%, mp: 210-214 °C; H NMR (400 MHz, DMSO-d6): δ 1.67-1.63(d, 2H, J=12.8), 2.01(br, s, 2H),
2.51-2.49(m, 2H, J=3.6), 3.44-3.38(t, 2H), 3.78-3.72(dd, 2H, J1=4.8, J2=12), 4.06-4.01(dd, 4H, J1=6.4, J2=12), 6.94-6.91(m, 2H),
7.09-7.04(m, 2H, J=2.4), 7.78(s, br, 1H, ex), 8.10(s, 1H, ex), 8.65(s, 1H, ex), 9.13(s, br, 1H, ex); HRMS calcd. for C16H22FN5O3
[M-HBr+H]+: 352.1779. found: 352.1784.
4.2.4 2,4-Diamino-5-(3'-(4''-methoxy-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A4)

ACCEPTED MANUSCRIPT
White solid yield: 48.5%, mp: 217-219 °C;HPLC: 99.78% (tR=7.026 min); 1H NMR (400 MHz, DMSO-d6): δ 1.69-1.66(d,
2H, J=12.8), 2.0w(br, s, 2H), 2.16-2.11(t, 2H, J=6), 3.55-3.49(t, 2H, J=11.8), 3.80-3.76(dd, 2H, J1=4.8, J2=12), 3.69(s, 3H),
4.07-4.00(m, 4H), 6.87-6.82(m, 4H), 7.85(s, br, 1H, ex), 8.09(s, 1H, ex), 8.67(s, 1H, ex), 9.11(s, 1H, ex); 13C NMR (151 MHz,
DMSO-d6): δ 161.60, 157.17, 134.59, 130.20, 129.39, 128.86, 79.61, 72.27, 62.71, 33.38, 32.08; HRMS calcd. for C17H25N5O4
[M-HBr+H]+: 364.1979. found: 364.1985.
4.2.5 2,4-Diamino-5-(3'-(4''-tert-butyl-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A5)
White solid yield: 65.8%, mp: 216-218 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.25(s,9H), 1.69-1.66(d,2H, J=12.8), 2.02(br,
s, 2H), 2.16-2.13(t, 2H, J=6), 3.52-3.46(t, 2H, J=12), 3.79-3.75(dd, 2H, J1=4.4, J2=12), 4.07-4.04(t, 4H, J=6), 6.91-6.88(d, 2H,
J=9.2), 7.33-7.31(d, 2H, J=8.8), 7.85(s, br, 1H, ex), 8.10(s, 1H, ex), 8.67(s, 1H, ex), 8.92(s, 1H, ex); 13C NMR (151 MHz,
DMSO-d6): δ 161.60, 157.17, 134.59, 130.20, 129.39, 128.86, 79.61, 72.27, 62.71, 33.38, 32.08; HRMS calcd. for C20H31N5O3
[M-HBr+H]+: 390.2500. found: 390.2499.
4.2.6 2,4-Diamino-5-(3'-(4''-chrolo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A6)
1
White solid yield: 23.9%, mp: 217-219 °C; H NMR (400 MHz, DMSO-d6): δ 1.68-1.65(d,2H, J=12.8), 2.00(br, s, 2H),
2.15-2.12(t, 2H, J=6), 3.47-3.41(t, 2H, J=12), 3.78-3.74(dd, 2H, J1=4.8, J2=12), 4.08-4.04(dd, 4H, J1=6, J2=12), 6.91-6.88(d, 2H,
J=8.8), 7.31-7.29(d, 2H, J=8), 7.80(s, br, 1H, ex), 8.11(s, 1H, ex), 8.65(s, 1H, ex), 8.90(s, 1H, ex); 13C NMR (151 MHz, DMSO-
d6): δ 161.43, 157.60, 156.94, 129.74, 124.81, 116.77, 74.95, 72.26, 64.83, 62.65, 31.98, 27.35; HRMS calcd. for C16H22ClN5O3
[M-HBr+H]+: 368.1484. found: 368.1485.
4.2.7 2,4-Diamino-5-(3'-(4''-methyl-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A7)
1
White solid yield: 62.1%, mp: 221-222 °C; H NMR (400 MHz, DMSO-d6): δ 1.70-1.67(d, 2H, J=12), 2.03(br, s, 2H),
2.16-2.13(t, 2H, J=6), 2.23(s,3H), 3.52-3.46(t, 2H, J=12), 3.80-3.76(dd, 2H,J1=4.4, J2=12), 4.08-4.03(dd, 4H, J1=6, J2=12), 6.82-
6.80(d, 2H, J=8.8), 7.08-7.05(d, 2H, J=8.4), 7.85(s, br, 1H, ex), 8.10(s, 1H, ex), 8.68(s, 1H, ex), 9.05(s, 1H, ex); 13C NMR (151
MHz, DMSO-d6): δ 161.49, 156.96, 156.61, 130.31, 129.79, 114.81, 75.12, 72.22, 64.39, 62.68, 31.75, 27.50, 20.55; HRMS
calcd. for C17H25N5O3 [M-HBr+H]+: 348.2030. found: 348.2037.
4.2.8 2,4-Diamino-5-(3'-(2'',4'',5''-trichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide
(A8)
1
White solid yield: 45.2%, mp: 215-217 °C; HPLC: 98.93% (tR=15.967 min); H NMR (400 MHz, DMSO-d6): δ 1.69-
1.66(d, 2H.J=12.8), 2.01(br, s, 2H), 2.22-2.19(t, 2H, J=6.4), 3.53-3.46(t, 2H, J=12), 3.79-3.75(dd, 2H, J1=4.8, J2=11.8), 4.09-
4.06(t, 2H, J=6.4), 4.27-4.22(t, 2H, J=6), 7.49(s, 1H), 7.79(s, 1H), 6.91(s, br, 1H, ex), 8.12(s, 1H, ex), 8.68(s, 1H, ex), 9.03(s, 1H,
ex); 13C NMR (151 MHz, DMSO-d6): δ 161.54, 156.88, 153.61, 131.08, 130.98, 123.30, 121.63, 115.95, 74.56, 72.15, 66.35,
62.76, 32.01, 27.17; HRMS calcd. for C16H20Cl3N5O3 [M-HBr+H]+: 436.0704, found: 436.0708.
4.2.9 2,4-Diamino-5-(3'-(4''-bromo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A9)
1
White solid yield: 31.6%, mp: 215-216 °C; H NMR (400 MHz, DMSO-d6): δ 1.69-1.66(d, 2H, J=12.8), 2.01(br, s, 2H),
2.17-2.12(t, 2H, J=6), 3.54-3.48(t, 2H, J=12), 3.79-3.75( dd, 2H, J1=4.4,J2=12), 4.09-4.04(m, 4H), 6.92-6.89(d, 2H, J=8.8), 7.43-
7.41(d, 2H, J=8.4), 7.95(s, br, 1H, ex), 8.09(s, 1H, ex), 8.66(s, 1H, ex), 9.21(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ
161.47, 158.05, 157.03, 132.63, 117.28, 112.56, 74.95, 72.23, 64.78, 62.68, 31.84, 27.34; HRMS calcd. for C16H22BrN5O3 [M-
HBr+H]+: 412.0979. found: 412.0976
4.2.10 2,4-Diamino-5-(3'-(3'',4''-dichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide
(A10)
1
White solid yield: 52.3%, mp: 218-220 °C; H NMR (400 MHz, DMSO-d6): δ 1.70-1.67(d, 2H, J=12.8), 2.01(br, s, 2H),
2.17-2.14(t, 2H, J=6), 3.54-3.48(t, 2H, J=12), 3.80-3.76(dd, 2H, J1=4.8, J2=12), 4.07-4.04(t, 2H, J=6), 4.14-4.11(t, 2H, J=6),
7.00-6.97(dd, 1H, J1=2.8, J2=9.2), 7.25-7.24 (d, 1H, J=2.8), 7.53-7.51(m, 1H), 7.98(s, br, 1H, ex), 8.11(s, 1H, ex), 8.68(s, 1H,
ex), 9.08(s, 1H, ex); HRMS calcd. for C16H21Cl2N5O3 [M-HBr+H]+: 402.1094. found: 402.1101.
4.2.11 2,4-Diamino-5-(3'-(2'',3''-dichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide
(A11)
1
White solid yield: 30.7%, mp: 208-210 °C (dec) ; H NMR (400 MHz, DMSO-d6): δ 1.67-1.64 (d, 2H, J=12.4), 2.00(br, s,
2H), 2.23-2.20(t, 2H, J=6), 3.61-3.58(m, 2H), 3.73-3.69(dd, 2H, J1=4.8, J2=12), 4.11-4.08(t, 2H, J=6), 4.23-4.20(t, 2H, J=6),
7.23-7.17(m, 2H), 7.35-7.31(t, 2H, J=8), 7.81(s, br, 1H, ex), 8.10(s, 1H, ex), 8.65(s, 1H, ex), 9.65(s, 1H, ex); 13C NMR (151
MHz, DMSO-d6) : δ 161.60, 157.17, 134.59, 130.20, 129.39, 128.86, 79.61, 72.27, 62.71, 33.38, 32.08; HRMS calcd. for
C16H21Cl2N5O3 [M-HBr+H]+: 402.1094. found: 402.1100.
4.2.12 2,4-Diamino-5-(3'-(3'',5''-dimethyl-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide
(A12)

ACCEPTED MANUSCRIPT
White solid yield: 52.0%, mp: 203-205 °C; HPLC: 99.27% (tR=6.101 min); 1H NMR (400 MHz, DMSO-d6): δ 1.70-1.67 (d,
2H, J=12.8), 2.04-1.99(m, 2H), 2.13-2.16(m, 2H), 2.23(s, 6H), 3.56-3.50(t, 2H, J=12), 3.81-3.77(dd,2H, J1=4.4, J2=12), 4.08-
4.03(dd, 4H, J1=6, J2=10.8), 6.69-6.58(d,3H,J=8.4), 7.83(s, br, 1H, ex), 8.11(s, 1H, ex), 8.66(s, 1H, ex), 9.36(s, 1H, ex); 13C
NMR (151 MHz, DMSO-d6): δ 161.56, 158.76, 156.96, 139.09, 122.81, 112.73, 75.18, 72.15, 64.23, 62.74, 56.49, 31.76, 27.50,
21.52, 19.02; HRMS calcd. for C18H27N5O3 [M-HBr+H]+: 362.2187. found: 362.2193.
4.2.13 2,4-Diamino-5-(3'-(2''-chrolo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A13)
1
White solid yield: 37.7%, mp: 207-209 °C (dec); H NMR (400 MHz, DMSO-d6): δ 1.69-1.66(d, 2H, J=12.4), 2.01(br, s,
2H), 2.22-2.17(dd,2H, J1=6,J 2=12), 3.49-3.43(t, 2H, J=12), 3.76-3.72(dd,2H, J1=4.8, J2=12), 4.12-4.09(t, 2H, J=6), 4.19-4.16(t,2
H, J=6), 6.98-6.94(m, 1H), 7.18-7.16(dd, 1H, J1=1.6, J2=8), 7.33-7.28(m, 1H), 7.43-7.40(dd, 1H, J1=1.6, J2=8), 8.13(s, 1H, ex),
8.67(s, 1H, ex), 9.96(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.61, 156.87, 154.02, 130.38, 128.90, 122.14, 121.80,
114.44, 74.65, 72.03, 65.18, 62.75, 31.75, 27.38; HRMS calcd. for C16H22ClN5O3 [M-HBr+H]+: 368.1484. found: 368.1484.
4.2.14 2,4-Diamino-5-(3'-(3''-chrolo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A14)
1
White solid yield: 41.6%, mp: 209-211 °C; H NMR (400 MHz, DMSO-d6): δ 1.70-1.67(d,2H, J=13.2), 2.01(br, s, 2H),
2.17-2.14(t, 2H, J=6), 3.54-3.48(t, 2H, J=12), 3.80-3.76(dd,2H,J1=4.8,J2=12), 4.08-4.04(t,2H,J=6), 4.13-4.10(t,2H,J=6), 7.03-
6.92(m,3H), 7.33-7.29(t,1H,J=8), 8.11(s, 1H, ex), 8.68(s, 1H, ex), 9.05(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.61,
159.73, 156.87, 134.24, 131.40, 121.16, 115.05, 114.08, 74.98, 72.05, 64.91, 62.79, 31.78, 27.30; HRMS calcd. for
C16H22ClN5O3 [M-HBr+H]+: 368.1484. found: 368.1487.
4.2.15 2,4-Diamino-5-(3'-(2''-bromo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A15)
1
White solid yield: 38.0%, mp: 215-216 °C; H NMR (400 MHz, DMSO-d6): δ 1.70-1.60(d,2H, J=12.8), 2.01(br, s, 2H),
2.23-2.19(dd, 2H, J1=6, J2=12), 3.47-3.41(t, 2H, J=12), 3.75-3.71(dd, 2H,J1=4.8, J2=12), 4.13-4.10(t, 2H, J=6), 4.19-4.16(t, 2H,
J=6), 6.92-6.89(m, 1H), 7.15-7.13(d, 1H, J=8.4), 7.37-7.33(m, 1H), 8.13(s, 1H, ex), 8.67(s, 1H, ex), 8.89(s, br, 1H, ex); 13C
NMR (151 MHz, DMSO-d6): δ 161.64, 156.85, 154.86, 133.41, 129.59, 122.66, 114.28, 111.45, 74.63, 71.99, 65.15, 62.76,
31.91, 27.39; HRMS calcd. for C16H22BrN5O3 [M-HBr+H]+: 412.0979. found: 412.0983.
4.2.16 2,4-Diamino-5-(3'-(3''-bromo-phenoxy)propyloxy)-1,3,5-triaza-9-oxa-spiro[5.5]undeca-1,3-diene hydrobromide (A16)
White solid yield: 56.6%, mp: 210-212 °C; HPLC: 98.54% (tR=11.398 min); 1H NMR (400 MHz, DMSO-d6): δ 1.70-
1.67(d, 2H, J=12.8), 2.02(br, s, 2H), 2.19-2.13(t, 2H, J=6), 3.53-3.47(t, 2H, J=12), 3.80-3.77(dd, 2H, J1=4.4, J2=12), 4.13-
4.04(m, 4H), 7.00-6.96(dd, 1H, J1=2.4, J2=8), 7.17-7.12(m, 2H), 7.27-7.23(m, 1H), 8.10(s, 1H, ex), 8.68(s, 1H, ex), 9.02(s, 1H,
ex); 13C NMR (151 MHz, DMSO-d6): δ 161.59, 159.77, 156.82, 131.73, 124.08, 122.60, 117.89, 114.46, 74.93, 72.17, 64.94,
62.83, 31.58, 27.32; HRMS calcd. for C16H22BrN5O3 [M-HBr+H]+: 412.0979. found: 412.0982.
4.3 General Procedure for the Synthesis of B1-B12 series
The mixture of substituted phenoxypropyloxy hydroxylamine hydrochlorides (0.005 mol), dicyandiamide (0.4 g, 0.005
mol) and EtOH (25 ml) was refluxed for 5h, follwed by being cooled to room temperature. To a suspension of the resulting
biguanide hydrochlorides in absolute EtOH (25 ml) was added conc. HCl (0.15 ml), was stirred at room temperature for 4 days
to 15 days. After evaporation of the solvent, the residue was triturated with acetone. The solid was collected by filtration and
washed again with ether and dried under oven. All samples were recrystallized from ethanol-water before analysis.
4.3.1 2,4-Diamino-5-(3'-(4''-bromo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B1)
1
White solid yield: 52.9%, mp: 212-214 °C (dec); H NMR (400 MHz, DMSO-d6): δ 2.03-2.00(m, 4H), 2.17-2.14(t, 2H,
J=6), 2.77-2.67(m, 2H), 2.88(br, s, 2H), 4.10-4.00(dd, 4H, J1=6, J2=12.8), 6.95-6.89(dd, 2H, J1=4.4, J2=14.4), 7.46-7.41(t, 2H,
J=8.8), 7.88(s, br, 1H, ex), 8.08(s, 1H, ex), 8.66(s, 1H, ex), 9.14(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.28, 158.08,
156.86, 132.63, 117.30, 112.57, 74.87, 73.80, 64.70, 27.36, 23.61; HRMS calcd. for C16H22BrN5O2S [M-HCl+H]+: 428.0750.
found: 428.0745
4.3.2 2,4-Diamino-5-(3'-(4''-chrolo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B2)
1
White solid yield: 25.5%, mp: 226-228 °C (dec); HPLC: 99.93% (tR=11.658 min); H NMR (400 MHz, DMSO-d6): δ
2.06(s, 4H), 2.16-2.13(t, 2H, J=6), 2.52-2.50(m, 2H), 2.83(br, s, 2H), 4.09-4.04(m, 4H), 7.00-6.96(m, 2H), 7.33-7.29(m, 2H),
8.07(s, 1H, ex), 8.62(s, 1H, ex), 8.94(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.25, 157.64, 156.92, 129.72, 124.86,
116.76, 74.86, 73.86, 64.75, 27.38, 23.62; HRMS calcd. for C16H22ClN5O2S [M-HCl+H]+: 384.1255. found: 384.1256.
4.3.3 2,4-Diamino-5-(3'-(2'',4''-dichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B3)
1
White solid yield: 45.3%, mp: 210-212 °C (dec); HPLC: 99.81% (tR=12.855 min); H NMR (400 MHz, DMSO-d6): δ
2.05(s, 4H), 2.20-2.19(t, 2H, J=6), 2.49-2.44(m, 2H), 2.89(br, s, 2H), 4.11-4.08(t, 2H, J=6), 4.20-4.17(t, 2H, J=6), 7.21-7.19(d,
1H, J=8.8), 7.39-7.36(dd,1H, J1=2.4, J2=8.8), 7.56-7.55(d, 1H, J=2.4), 7.82(s, br, 1H, ex), 8.10(s, 1H, ex), 8.66(s, 1H, ex), 9.23(s,
1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.28, 156.85, 153.21, 129.71, 128.69, 125.07, 122.81, 115.59, 74.40, 73.83,
65.58, 27.30, 23.63; HRMS calcd. for C16H21Cl2N5O2S [M-HCl+H]+: 418.0866. found: 418.0871.

ACCEPTED MANUSCRIPT
4.3.4 2,4-Diamino-5-(3'-(4''-methoxy-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B4)
1
White solid yield: 58.6%, mp: 220-222 °C; H NMR (400 MHz, DMSO-d6): δ 2.07(s, 4H), 2.15-2.12(t, 2H, J=6), 2.55-
2.50(m, 2H), 2.87(br, s, 2H), 3.69(s,3H), 4.09-4.02(m,4H), 6.91-6.85(m,4H), 8.07(s, 1H, ex), 8.67(s, 1H, ex); 13C NMR (151
MHz, DMSO-d6): δ 161.26, 156.93, 153.97, 152.76, 115.96, 115.11, 75.04, 73.86, 64.87, 55.87, 27.59, 23.61; HRMS calcd. for
C17H25N5O3S [M-HCl+H]+: 380.1751. found: 380.1750.
4.3.5 2,4-Diamino-5-(3'-(4''-tert-butyl-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B5)
1
White solid yield: 57.3%, mp: 232-234 °C; HPLC: 99.71% (tR=14.986 min); H NMR (400 MHz, DMSO-d6): δ 1.25(s,
9H), 2.05(s, 4H), 2.17-2.13(t, 2H, J=6), 2.50-2.45(m, 2H), 2.88(br, s, 2H), 4.09-4.06(t, 4H, J=6), 6.89-6.86(d, 2H, J=8.8), 7.30-
7.28(t, 2H, J=8.8), 8.07(s, 1H, ex), 8.68(s, 1H, ex), 9.18(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.27, 156.91, 156.54,
143.33, 126.55, 114.49, 74.93, 73.82, 64.25, 34.23, 31.82, 27.55, 23.57; HRMS calcd. for C20H31N5O2S [M-HCl+H]+: 406.2271.
found: 406.2274.
4.3.6 2,4-Diamino-5-(3'-(2''-chrolo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B6)
1
White solid yield: 25.5%, mp: 226-228 °C (dec); H NMR (400 MHz, DMSO-d6): δ 2.05(s, 4H), 2.22-2.19(t, 2H, J=6),
2.45-2.42(d, 2H, J=13.6), 2.91(br, s, 2H), 4.20-4.10(m, 4H), 6.98-6.94(m, 1H), 7.18-7.16(m, 1H), 7.32-7.29(m, 1H), 7.43-
7.40(m, 1H), 7.84(s, br, 1H, ex), 8.10(s, 1H, ex), 8.65(s, 1H, ex), 9.32(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.28,
156.89, 154.04, 130.35, 128.90, 122.13, 121.80, 114.44, 74.48, 73.83, 65.08, 27.42, 23.60; HRMS calcd. for C16H22ClN5O2S
[M-HCl+H]+: 384.1255. found: 384.1259.
4.3.7 2,4-Diamino-5-(3'-(3''-chrolo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B7)
1
White solid yield: 30.8%, mp: 209-211 °C; H NMR (400 MHz, DMSO-d6): δ 2.07(s, 4H), 2.18-2.15(t, 2H, J=6), 2.50-
2.47(d, 2H, J=12), 2.93(br, s, 2H), 4.14-4.06(m, 4H), 7.04-6.93(m, 3H), 7.33-7.29(t, 1H, J=8), 7.85(s, br, 1H, ex), 8.08(s, 1H,
ex), 8.66(s, 1H, ex), 9.27(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.26, 159.75, 156.91, 134.25, 131.37, 121.14, 115.06,
114.11, 74.81, 73.85, 64.80, 27.34, 23.62; HRMS calcd. for C16H22ClN5O2S [M-HCl+H]+: 384.1255. found: 384.1261.
4.3.8 2,4-Diamino-5-(3'-(2''-bromo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B8)
1
White solid yield: 41.6%, mp: 214-216 °C; H NMR (400 MHz, DMSO-d6): δ 2.06(s, 4H), 2.22-2.19(t, 2H, J=6), 2.46-
2.42(d, 2H, J=14), 2.89(br, s, 2H), 4.20-4.12(m, 4H), 6.93-6.89(m, 1H), 7.16-7.14(d, 1H, J=8.4), 7.37-7.33(m, 1H), 7.59-7.57(m,
1H), 7.86(s, br, 1H, ex), 8.12(s, 1H, ex), 8.66(s, 1H, ex), 9.22(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.27, 156.90,
154.87, 133.37, 129.57, 122.64, 114.29, 111.46, 74.45, 73.83, 65.06, 27.44, 23.60; HRMS calcd. for C16H22BrN5O2S [M-
HCl+H]+: 428.0750. found: 428.0747.
4.3.9 2,4-Diamino-5-(3'-(3''-bromo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride (B9)
1
White solid yield: 22.5%, mp: 210-212 °C; H NMR (400 MHz, DMSO-d6): δ 2.07(s, 4H), 2.17-2.14(t, 2H, J=6), 2.50-
2.47(d, 2H, J=14), 2.91-2.93(d, 2H, J=5.6), 4.14-4.06(m, 4H), 7.00-6.97(dd, 1H, J1=2.4, J2=8), 7.18-7.12(m, 2H), 7.27-7.23(t,
1H, J=8), 7.84(s, br, 1H, ex), 8.08(s, 1H, ex), 8.66(s, 1H, ex), 9.25(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.25,
159.79, 156.92, 131.70, 124.07, 122.61, 117.92, 114.47, 74.81, 73.86, 64.82, 27.35, 23.63; HRMS calcd. for C16H22BrN5O2S
[M-HCl+H]+: 428.0750. found: 428.0744.
4.3.10 2,4-Diamino-5-(3'-(2'',3''-dichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride
(B10)
1
White solid yield: 50.3%, mp: 223-225 °C (dec); HPLC: 100.00% (tR=12.457 min); H NMR (400 MHz, DMSO-d6): δ
2.04(s, 4H), 2.23(br, s, 2H), 2.42-2.40(d, 2H, J=8), 2.92(br, s, 2H), 4.13-4.10(t, 2H, J=8), 4.23-4.21(t, 2H, J=4), 7.24-7.18(m,
2H), 7.37-7.32(t, 1H, J=8), 7.82(s, br, 1H, ex), 8.17(s, 1H, ex), 8.71(s, 1H, ex), 9.40(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6):
δ 161.26, 156.87, 155.63, 132.72, 129.13, 122.64, 120.50, 112.87, 74.38, 73.83, 65.79, 27.32, 23.61; HRMS calcd. for
C16H21Cl2N5O2S [M-HCl+H]+: 418.0866. found: 418.0869.
4.3.11 2,4-Diamino-5-(3'-(3'',4''-dichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride
(B11)
1
White solid yield: 38.7%, mp: 229-231 °C; HPLC: 99.28% (tR=13.119 min); H NMR (400 MHz, DMSO-d6): δ 2.06(s,
4H), 2.16(br, s, 2H), 2.51-2.50(m, 2H), 2.94(br, s, 2H), 4.14-4.11(m, 4H), 7.02-6.99(dd, 1H, J1=4, J2=8), 7.28-7.27(d, 1H, J=4),
7.54-7.52(d, 1H, J=8), 7.82(s, br, 1H, ex), 8.13(s, 1H, ex), 8.70(s, 1H, ex), 9.44(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ
161.26, 158.30, 156.89, 132.11, 131.46, 123.03, 116.95, 116.02, 74.73, 73.85, 65.29, 27.25, 23.64; HRMS calcd. for
C16H21Cl2N5O2S [M-HCl+H]+: 418.0866. found: 418.0866.
4.3.12 2,4-Diamino-5-(3'-(2'',4'',5''-trichrolo-phenoxy)propyloxy)-1,3,5-triaza-9-thia-spiro[5.5]undeca-1,3-diene hydrochloride
(B12)
1
White solid yield: 23.9%, mp: 220-222 °C (dec); H NMR (400 MHz, DMSO-d6): δ 2.07(s, 4H), 2.24(br, s, 2H), 2.52-
2.47(m, 2H), 2.96(br, s, 2H), 4.17-4.13(m, 4H), 7.37(s, 1H), 7.67(s, 1H), 7.87(s, br, 1H, ex), 8.12(s, 1H, ex), 8.67(s, 1H, ex),

ACCEPTED MANUSCRIPT
9.17(s, 1H, ex); 13C NMR (151 MHz, DMSO-d6): δ 161.28, 156.84, 153.65, 130.99, 123.33, 121.62, 115.98, 74.29, 73.84, 69.49,
66.18, 33.53, 29.16, 27.17, 23.67; HRMS calcd. for C16H20Cl3N5O2S [M-HCl+H]+: 452.0476. found: 452.0482.
Acknowledgments
This work was sponsored by Natural Science Foundation of Shanghai (No. 15ZR1440200). We gratefully appreciate the
excellent assay assistance from Mrs. Chen Xiu Hua and Mrs. Wang Juan, Shanghai Institute of Pharmaceutical Industry.
References
[1] Berman, E. M., & Werbel, L. M. (1991). The renewed potential for folate antagonists in contemporary cancer chemotherapy. Journal of Medicinal Chemistry,
34(2), 479-485.
[2] Huang, H., Lu, W., Xi, L., Cong, X., Ma, H., & Liu, X., et al. (2011). Design and synthesis of small molecular dual inhibitor of falcipain-2 and dihydrofolate
reductase as antimalarial agent. Bioorganic & Medicinal Chemistry Letters, 22(2), 958-962.
[3] Kompis, Ivan M., Islam, Khalid, & Then, Rudolf L. (2005). Dna and rna synthesis: antifolates. Chemical Reviews, 105(2), 593-620.
[4] Gangjee, A., Jain, H. D., & Kurup, S. (2007). Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents:
part ii. Anti-cancer agents in medicinal chemistry, 7(5), 524-542.
[5] Kisliuk, R. L. (2003). Deaza analogs of folic acid as antitumor agents. Current Pharmaceutical Design, 9(31), 2615-2625.
[6] Mcguire, J. J. (2003). Anticancer antifolates: current status and future directions. Current Pharmaceutical Design, 9(31), 2593-2613.
[7] Hanauske, A. R., Chen, V., Paoletti, P., et al. (2001). Pemetrexed disodium: a novel antifolate clinically active against multiple solid tumors. Oncologist,
6(4), 363-373.
[8] Punt, C. J. (2000) Trimetrexate as a biochemical modulator of 5-fluorouracil and leucovorin in colorectal cancer. Seminars in Oncology, 27(10), 88-90.
[9] Tomasini, P., Barlesi, F., Mascaux, C., et al. (2016) Pemetrexed for advanced stage nonsquamous non-small cell lung cancer: latest evidence about its
extended use and outcomes. Therapeutic Advances in Medical Oncology, 8(3), 198-208.
[10] Rosowsky, A., Mota, C. E., Queener, S. F. (2012). ChemInform Abstract: Synthesis and Antifolate Activity of 2,4-Diamino-5,6,7,8- tetrahydropyrido(4,3-
d)pyrimidine Analogues of Trimetrexate and Piritrexim. Journal of Heterocyclic Chemistry, 26(30), 335-340.
[11] Gonen, N., Assaraf, Y. G. (2012). Antifolates in cancer therapy: Structure, activity and mechanisms of drug resistance. Drug Resistance Updates Reviews
& Commentaries in Antimicrobial & Anticancer Chemotherapy, 15(4), 183-210.
[12] Cody, V., Luft, J.R., Pangborn, W.,(2005). Understanding the role of Leu22 variants in methotrexate resistance: comparison of wild-type and Leu22Arg
variant mouse and human dihydrofolate reductase ternary crystal complexes with methotrexate and NADPH, Acta Crystallographica Section D 61(2), 147-
155.
[13] Ma, X., Woon, S. P., Ho, C. L., & Chui, W. K. (2009). Antiproliferative activity against mcf-7 breast cancer cells by diamino-triazaspirodiene antifolates.
Chemical Biology & Drug Design, 74(3), 322-326.
[14] Ma, X., Chui, W. K. (2010) Antifolate and antiproliferative activity of 6,8,10-triazaspiro(4.5)deca-6,8-dienes and 1,3,5-triazaspiro(5.5)undeca-1,3-dienes,
Bioorganic & Medicinal Chemistry, 18(2), 737-743.
[15] Yuthavong, Y., Tarnchompoo, B., Vilaivan, T., Chitnumsub, P., Kamchonwongpaisan, S., & Charman, S. A., et al. (2012). Malarial dihydrofolate reductase
as a paradigm for drug development against a resistance-compromised target. Proceedings of the National Academy of Sciences of the United States of
America, 109(42), 16823-16828.
[16] William, S. L., Cody, V., Galitsky N., Luft, J. R, Pangborn, W., & Chunduru, S. K., et al. (1995). Methotrexate-resistant variants of human dihydrofolate
reductase with substitutions of leucine 22. Journal of Biological Chemistry, 270(10), 5057-64.
[17] Klon, A. E., Héroux, A., Ross, L. J., Pathak, V., Johnson, C. A., & Piper, J. R., et al. (2002). Atomic structures of human dihydrofolate reductase
complexed with nadph and two lipophilic antifolates at 1.09Å and 1.05Å resolution. Journal of Molecular Biology, 320(3), 677-693.
[18] Cody, V., Galitsky N., Luft, J. R, Pangborn ,Walter., Rosowsky, A., Blakley, R. L. (1997). Comparison of two independent crystal structures of human
dihydrofolate reductase ternary complexes reduced with nicotinamide adenine dinucleotide phosphate and the very tight-binding inhibitor pt523,
Biochemistry, 36(45), 13897-13903.
[19] Cody, V., Luft, J. R., Pangborn, W., Gangjee, A., & Queener, S. F. (2001). Structure determination of tetrahydroquinazoline antifolates in complex with
human and dihydrofolate reductase: correlations between enzyme selectivity and stereochemistry. Acta Crystallographica, 60(4):646–655.
[20] Volpato, J. P., Yachnin, B. J., Blanchet, J., Guerrero, V., Poulin, L., & Fossati, E., et al. (2009). Multiple conformers in active site of human dihydrofolate
reductase f31r/q35e double mutant suggest structural basis for methotrexate resistance. Journal of Biological Chemistry, 284(30), 20079-20089.
[21] Cody, V., & Pace, J. (2011). Structural analysis of pneumocystis carinii, and human dhfr complexes with nadph and a series of five potent 6-[5’-(ω-
carboxyalkoxy)benzyl]pyrido[2,3- d ]pyrimidine derivatives. Acta Crystallographica, 67(Pt 1), 1-7.
[22] Chunduru, S. K., Cody, V., Luft, J. R., Pangborn, W., Appleman, J. R., & Blakley, R. L. (1994). Methotrexate-resistant variants of human dihydrofolate
reductase. effects of phe31 substitutions. Journal of Biological Chemistry, 269(13), 9547-9555.
[23] Cody, V., Piraino, J., Pace, J., Li, W., & Gangjee, A. (2001). Preferential selection of isomer binding from chiral mixtures: alternate binding modes
observed for the e and z isomers of a series of 5-substituted 2,4-diaminofuro[2,3- d]pyrimidines as ternary complexes with nadph and human dihydrofolate
reductase. Acta Crystallographica, 66(12), 1271-1277.
[24] Erickson, J. A., Jalaie, M., Robertson, D. H., Lewis, R. A., & Vieth, M. (2004). Lessons in molecular recognition: the effects of ligand and protein
flexibility on molecular docking accuracy. Journal of Medicinal Chemistry, 47(1), 45-55.
[25] Alberts, I. L., Todorov, N. P., & Dean, P. M. (2005). Receptor flexibility in de novo ligand design and docking. Journal of Medicinal Chemistry,48(21),
6585-6596.
[26] Koska, J., Spassov, V.Z.; Maynard, A.J.; Yan, L.; Austin, N.; Flook, P.K.; Venkatachalam, C.M. Fully automated molecular mechanics based induced fit
protein-ligand docking method, Journal of Chemical Information and Modeling, 2008, 48(10), 1965-1973.
[27] Adamson, P. C., Balis, F. M., Mccully, C. L., Godwin, K. S., & Poplack, D. G. (1992). Methotrexate pharmacokinetics following administration of
recombinant carboxypeptidase-g2 in rhesus monkeys. Journal of Clinical Oncology, 10(8), 1359-1364.
[28] Pignatello, R., Spampinato, G., Sorrenti, V., Vicari, L., Giacomo, C. D., & Vanella, A., et al. (1999). Aliphatic ɑ,γ-bis(amides) of methotrexate. influence
of chain length on in-vitro activity against sensitive and resistant tumour cells. Pharmacy & Pharmacology Communications, 5(4), 299-305.
[29] Grever, M. R., Schepartz, S. A., & Chabner, B. A. (1993). The national cancer institute: cancer drug discovery and development program. Seminars in
Oncology, 19(6), 622-638.
[30] Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull, K., & Vistica, D., et al. (1991). Feasibility of a high-flux anticancer drug screen using a diverse
panel of cultured human tumor cell lines. Journal of the National Cancer Institute, 83(11), 757-766.
[31] Liu, X., Liu, J., Guan, Y., Li, H., Huang, L., & Tang, H., et al. (2012). Establishment of an orthotopic lung cancer model in nude mice and its evaluation by
spiral ct. Journal of Thoracic Disease, 4(2), 141-145.
[32] Cheng, X., Zhou, T., Li B., & Li, M., et al. (2013). Methotrexate and 5-aminoimidazole-4-carboxamide riboside exert synergistic anticancer action against
human breast cancer and hepatocellular carcinoma. Acta Pharmacologica Sinica, 34(7), 951-959.

ACCEPTED MANUSCRIPT
Table 1 in vitro hDHFR inhibitory activity and in vitro anti-proliferative activity
hDHFR
hDHFR
IC50a
(nM)
Anti-proliferative activity IC50(μM)
Inhibition (%)
No
X
R
at
at
MDA-MB
-231
HCT116
A549
HL-60
HepG2
-
50µM
5µM
b
A1
A2
O
O
-
>90
>90
86.11
7.46
-
6.01
1.08
2.46
-
-
-
-
-
0.88
3.28
0.07
1.06
0.33
2.38
A3
A4
A5
O
O
O
>90
>90
>90
46.62
11.07
3.72
-
-
-
-
-
-
-
1.5
0.66
0.5
1.67
0.87
O
1.61
A6
A7
O
O
-
-
>90
>90
33.56
56.75
-
-
-
-
0.69
2.39
0.36
1.06
0.33
2.58
A8
O
-
>90
36.01
-
-
2.75
0.68
2.36
A9
O
O
-
-
>90
>90
32.49
8.23
-
-
0.32
0.09
0.28
0.79
0.31
1.24
A10
0.38
0.59
A11
O
89.8
81.7
-
0.005
0.1
0.44
0.33
0.14

ACCEPTED MANUSCRIPT
A12
O
88.6
84.3
-
0.19
0.71
0.72
0.68
3.57
A13
A14
A15
O
O
O
88.2
85.8
85.0
83.3
83.2
83.8
-
-
-
0.07
0.12
0.03
2.7
0.15
0.16
0.66
0.52
0.61
1.75
0.15
0.86
1.43
1.02
0.91
A16
B1
O
S
85.8
-
86.7
>90
-
0.04
0.02
0.16
0.74
0.56
0.35
0.32
1.4
0.4
6.46
0.44
B2
S
-
>90
18.28
0.01
0.58
0.43
1.1
0.35
B3
B4
B5
S
S
S
-
>90
81.3
85.3
4.08
0.001
0.07
0.21
0.24
0.33
0.5
1.38
0.88
0.07
0.06
0.35
O
86.6
86.6
-
-
O
0.001
0.001
0.28
0.001
B6
B7
B8
S
S
S
89.6
88.8
89.6
86.3
88.3
88.7
-
-
-
0.001
0.001
0.001
0.08
0.06
0.04
0.39
0.26
0.18
0.08
0.09
0.09
0.08
0.13
0.01
B9
S
S
S
86.5
89.8
89.8
84.2
83.8
88.4
-
-
-
0.001
0.001
0.001
0.05
0.001
0.001
0.2
0.03
0.14
0.09
0.05
0.07
0.06
0.001
0.01
B10
B11

ACCEPTED MANUSCRIPT
B12
M0
S
94.4
84.4
-
0.001
0.38
>100
0.99
0.73
0.58
>100
0.11
C
87.6
89.7
86.4
86.4
49.04
2.33
1.21
MTX
Doxorubicin
6.67
-
0.75
0.31
0.25
0.12
1.09
0.41
0.02
9.49
0.72
-
-
(a) Values are means of three experiments. (b)The symbol ‘-’ indicates ‘not tested.’
Table 2. The anticancer effect of compound A2 in an established nude mice A549 model.
Dose Treatment
RTV,
±SD
x
Group
(mg/kg) regimen
d1
d4
d8
d11
d15
4.28±0.64
d18
d22
7.77±1.20
Control
MTX
1.00±0.00
1.76±0.46
2.46±0.46
3.19±0.54
5.90±0.94
1.33±0.11* 1.65±0.23** 1.94±0.23** 2.36±0.36** 2.91±0.42** 3.82±0.60**
2
ip×14
ip×14
1.00±0.00
1.00±0.00
(24.65%)a
(32.69%)
(29.19%)
(44.97%)
(50.65%)
(50.84%)
1.45±0.18
2.10±0.36
2.67±0.46 3.22±0.54** 3.97±0.75** 4.91±0.66**
A2
50
(17.58%)
(14.50%)
(16.34%)
(24.73%)
(32.81%)
(36.80%)
*P<0.05 versus control，**P<0.01 versus control, (a)value in the () was tumor growth inhibition rate
Table 3. The change in body weight of the nude mice
Gose Treatment
Body weight (g),
d11
x
±SD
Group
(mg/kg) regimen
d1
d4
d8
d15
25.41±1.04
d18
d22
25.59±1.16
21.40±
2.79**
Control
MTX
24.73±1.33
25.28±0.97
24.84±1.22
25.47±0.99
25.19±1.38 24.99±0.97
25.66±1.16
2
ip×14
ip×14
24.88±0.82 23.98±0.58* 21.85±2.23** 21.62±2.87**
A2
50
23.65±1.01
23.53±1.23 23.17±1.14* 22.97±2.28*
23.63±2.13
23.65±1.98* 23.47±1.96*
*P<0.05 versus control，**P<0.01 versus control,

ACCEPTED MANUSCRIPT
Figure 1. Structures of reported DHFR inhibitors.

ACCEPTED MANUSCRIPT
Figure 2. The conformations of PHE31 in 1U72 (white) and in
3NXV (purple).

ACCEPTED MANUSCRIPT
Figure 3. (A)The binding mode of MTX (grey) with hDHFR (PDB
ID: 1U72), (B)The docking pose of compound M0 (purple) with
hDHFR compared with the binding mode of MTX (yellow) in
X-ray (PDB ID: 1U72), (C) Molecular surface of active-site in MTX
(yellow) in X-ray, (D) Molecular surface of active-site in M0
(purple)-hDHFR docking result

ACCEPTED MANUSCRIPT
Figure 4. The docking result of compound A2 with hDHFR
(1U72)

ACCEPTED MANUSCRIPT
NH2
O
NH
NH2
N
OH
O
N
N
N
O
HCl
H2N
NH2
H
N
HCl
O
g
e
f
A0
NH2
N
O
O
N
HO
a
Br
O
R
R
R
H2N
N
O
b
HBr
O
OH
N
H
c
A1-A16
O
O
O
N
R
H
d
NH2
O
O
O
NH
NH2
N
N
N
R
N
S
O
O
H2N
HCl
H
H2N
N
R
S
HCl
e
f
C1-C12
B1-B12
Scheme 1. Synthesis of A1-A16 and B1-B12. Reagents and
conditions: (a) dibromoalkane, K2CO3, CH3CN, reflux, 8 h; (b)
NaOH, DMF, 8-24 h; (c) NaOH, EtOH, reflex 10 h; (d) conc. HCl,
EtOH, reflex 5 h; (e) EtOH, reflex 5-10h; (f) conc. HCl, ethanol,
rt. ; (g) 10% Pb/C, 90% EtOH, 1MPa, rt.

ACCEPTED MANUSCRIPT
Highlights
1. A novel series of dihydro-1,3,5-triazine derivatives bearing a heteroatom spi
ro-ring were designed and synthesized.
2. Docking studies showed the binding mode of triazaspirodiene derivatives to
inhibit hDHFR.
3 Compound A2, A5, B1, and B3 had potent inhibitory activites against hDHF
R, which was superior to MTX and the leading compound.
4. 24 Compounds showed anti-tumor activities toward several tumor cell lines
with IC50 values ranging from 0.79 to 0.001 µM, which was superior to MT
X.